W1193: Locoweed and its Fungal Endophyte: Impact, Ecology, and Management

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[12/15/2015] [01/04/2017] [11/28/2017] [11/28/2017] [12/11/2018] [12/11/2019]

Date of Annual Report: 12/15/2015

Report Information

Annual Meeting Dates: 11/10/2015 - 11/10/2015
Period the Report Covers: 10/01/2014 - 09/01/2015

Participants

Cook, Daniel (Daniel.cook@usda.ars.gov.us) – USDA/ARS Poisonous Plant Lab, Logan, UT;
Baucom, Deana (dbaucom@nmsu.edu) – New Mexico State University;
Keith, Barbara (bkeith@montana.edu) – Montana State University;
Schardl, Christopher (chris.schardl@uky.edu) – University of Kentucky;
Sterling, Tracy (tracy.sterling@montana.edu) – Montana State University;
Creamer, Rebecca (creamer@nmsu.edu) – New Mexico State University;
Cibils, Andres (acibils@nmsu.edu) – New Mexico State University;
Lehnhoff, Erik (lehnhoff@nmsu.edu) – New Mexico State University;
Noor, Aziza (anoor@nmsu.edu) – New Mexico State University;
Neyaz, Marwah – Dept EPPWS, New Mexico State University;
Appling, Talinna – Dept. LRES, Montana State University;

Brief Summary of Minutes

W1193 Locoweed Regional Project

Locoweed and its fungal endophyte: impact, ecology, and management

Period Covered: July 1, 2015 – December 31, 2015

Date of This Report:

Annual Meeting Date: November 10, 2015, in Las Cruces, NM

Participants/Affiliations:

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

Deana Baucom – Dept EPPWS, New Mexico State University, dbaucom@nmsu.edu

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

Andres Cibils – Animal and Range Science, New Mexico State University, acibils@nmsu.edu

Erik Lehnhoff – Dept EPPWS, New Mexico State University, lehnhoff@nmsu.edu

Aziza Noor – Molecular Biology, New Mexico State University, anoor@nmsu.edu

Marwah Neyaz – Dept EPPWS, New Mexico State University

Talinna Appling – Dept. LRES, Montana State University,

David Thompson – AES, New Mexico State University, dathomps@nmsu.edu

Summary of Minutes of Annual Meeting:

The group was welcomed by the project coordinator, David Thompson, who is also the Agricultural Experiment Station Director at New Mexico State University. He explained how regional projects work and the various potential roles for project participants.

Daniel Cook presented the history and background of locoweeds and locoism. De Soto and other Spanish explorers are thought to have been the first to notice locoism when their horses were poisoned while they explored the southwestern USA. Animals need to graze around 3 week prior to the onset of clinical symptoms. There are recent reports of cattle poisoning (of over 500 head of cattle) near Pueblo, CO, from consumption of Astragalus mollissimus. Animal susceptibility to swainsonine consumption has been experimentally determined; goats and horses are the most sensitive, followed by sheep and cows. Rats, mice and other small animals are significantly less sensitive to locoweed feeding. With the recent precipitation, higher populations of locoweeds are predicted in New Mexico and Texas. Animals tend to eat locoweeds in the fields in early spring and late fall, which is before and after grass is available for grazing. Swainsonine causes a lysosomal storage disease. Swainsona canescens was the plant from which swainsonine was originally isolated. Daniel’s lab and collaborators have identified an endophyte tentatively called Undifilum canescens from the plants. Ipomoea carnea, which is in the morning glory family, has been shown to contain swainsonine and calystegines, both toxic components. The endophytic fungus that was isolated from this plant is a likely new genus and species from the Chaetothryiales. Sida carpinifolia, which is in the Malvaceae family and has been found in Brazil and Argentina, has also been found to contain swainsonine, however, no fungal endophyte has been identified yet.

Deana Baucom presented her research on identification and characterization of polyketide synthases (PKS) from Undifilum oxytropis. Using sequence alignment tools and the as yet unassembled genome of the fungus, she discovered 22 PKSs that had homology to other fungi. She found Type I PKS of high reducing, partially reducing, and low reducing types, NRPS types, and hybrid PKS-NRPS types. One PKS had very high amino acid identity with the melanin PKS from Alternaria. One NRPS had very high amino acid identity with a virulence factor found in Alternaria and other fungi. A PKS-NRPS hybrid had good homology with several Metarhizium species, and is speculated to function in swainsonine biosynthesis.

Barb Keith presented the research from the Sterling laboratory at Monatana State University. Locoweeds have been reported in Montana since the early 1900s, and are predominantly Oxytropis spp. such as O. sericea. Previous work had shown that swainsonine increased in plants with shortened photoperiod, i.e. fall and winter. When transplanted into a common garden, Oxytropis has been long lived, despite the very cold weather found in Bozeman. In contrast, Astragalus mollissimus, which is not normally found in the state, grows as an annual and appears very susceptible to freeze damage. There were no differences in the survival rate of Oxytropis plants that contained (+) or did not contain (-) the Undifilum endophyte. There were also no differences between the E+ and E- plants in terms of transpiration, photosynthesis, or stomatal conductance, when testing flowering plants in June and July over a several year period. Studies to determine if endophyte presence helped A. mollissimus survive stress (transplanting) and cold temperatures, showed mixed results, with overall non-significant changes in photosynthesis. In 2014 and 2015, there were mixed results, but overall non-significant differences between E+ and E- plants in flowers/stem, pods/stem, and seeds/pod, and the overall germination, and germination rate.

Daniel Cook presented a research update of the recent work from his lab. He presented an extensive list of Astragalus species that were reported to contain swainsonine and his results when testing from both fresh and herbaria specimens. Around nine of the species tested did not contain swainsonine; the discrepancy was attributed to incorrect identification of the plant or collecting the incorrect plant or to low sensitivity in previous testing methods. A few plants had very low levels of swainsonine and a few had only 1 plant out of a large number that tested positive for swainsonine. Swainsonine identification was correlated with the South American Clade G group of Astragalus sp. and with the A. allochrous group. His lab has found swainsonine in many Ipomoea species, although the endophytes have not been characterized yet. He cooperated on a study looking at the effect of elevated CO₂ and endophyte presence on A. mollissimus growth and crude protein. E- plants with elevated CO₂ grew large than E+ plants, but crude proteins were lower in E- plants. No difference was found in swainsonine with elevated CO₂.

Chris Schardl discussed seed-transmitted endophytic fungi, primarily those from grasses, Ipomoea, and locoweeds. The fungi are diverse ascomycetes and most are cryptic and nonpathogenic. For grasses and locoweeds, the endophytes grow between cells. Grasses have endobiotic (Epichloe) endophytes and epibiotic (Atkinsonella and some Balansia) endophytes. Some Ipomoea species can have epibiotic Periglandula spp. endophytes, and others have endophytes of the fungal order Chaetothyriales which may also be epibiotic. Locoweeds have endobiotic (Undifilum) endophytes. Epichloe has different interactions with seed pre and post pollination. Before pollination, the mycelia goes through the placental pore into the ovule, but not into the embryo sac. After pollination, the mycelia goes throughout the embryo sac. Epichloe infects the anthers but not the pollen grains. Periglandula grows on the outside of Ipomoea leaves. It gets into the ovules of young plants and can be found associated with capsule walls, seed coats, and embryos of older plants. Periglandula can be found between tissue types associated with the surface of tissues, for example between leaf primordial.

Rebecca Creamer gave an overview of work completed by her former graduate student, Mohammad Alhawatema on Slafractonia leguminicola. The fungus, previously known as Rhizoctonia leguminicola, was demonstrated to be morphologically different from Rhizoctonia sp. It also has ITS sequence and gpd sequence that were closest to Pleiochaeta setosa, an ascomycete. The genus was renamed to Slafractonia. The polyketide synthase gene associated with melanin biosynthesis was identified based on homology with other ascomycetes and the gene sequenced. The sequence was used for RNAi using the pSilent vector. After introduction into fungal protoplasts, the resulting silenced colonies were light in color, moving to a light gray color, compared to the black wild type cultures. The lighter colored colonies had less expression of the melanin gene and had reduced swainsonine concentration compared to the wild type colonies.

The group discussed cooperative research projects and resources that could be shared among the group. Daniel Cook, Chris Schardl and Rebecca Creamer will collaborate on identification of the key genes in swainsonine biosynthesis. Barb Keith volunteered E+ and E- O. sericea plants from her common garden that were collected at the young plant stage and frozen. Daniel Cook has a field of about 500 O. sericea plants that were marked as E+ or E- that he will share.

There were several unanswered questions that were identified that need research. These included why A. thompsonii appears to be resistant to infection and some plants had very little infection. Does Undifilum play a role in pathogen defense, since it appears to be negatively correlated with pathogen presence? Can the plant/fungus interactions be better defined through RNA seq? What drives population level differences in infection?

Accomplishments and Impacts:

We assembled a group of researchers that includes university and government-based individuals . The group met, discussed the current status of locoweeds, locoism and fungal endophytes. The group set priorities for research and collaborations in the coming year.

Daniel Cook made progress on which locoweeds produce swainsonine and will write the work for publication.

A collaborative group of Daniel Cook, Chris Schardl, and Rebecca Creamer made progress on identifying the key enzymes in the swainsonine biosynthetic pathway The work will now be written for publication. Four fungi that produce swainsonine were sequenced, although the genome assembly has not been completed.

Publications: None for the time period covered

Accomplishments

Daniel Cook presented the history and background of locoweeds and locoism. De Soto and other Spanish explorers are thought to have been the first to notice locoism when their horses were poisoned while they explored the southwestern USA.  Animals need to graze around 3 week prior to the onset of clinical symptoms.  There are recent reports of cattle poisoning (of over 500 head of cattle) near Pueblo, CO, from consumption of Astragalus mollissimus.  Animal susceptibility to swainsonine consumption has been experimentally determined; goats and horses are the most sensitive, followed by sheep and cows.  Rats, mice and other small animals are significantly less sensitive to locoweed feeding.  With the recent precipitation, higher populations of locoweeds are predicted in New Mexico and Texas.  Animals tend to eat locoweeds in the fields in early spring and late fall, which is before and after grass is available for grazing.  Swainsonine causes a lysosomal storage disease. Swainsona canescens was the plant from which swainsonine was originally isolated.  Daniel’s lab and collaborators have identified an endophyte tentatively called Undifilum canescens from the plants. Ipomoea carnea, which is in the morning glory family, has been shown to contain swainsonine and calystegines, both toxic components.  The endophytic fungus that was isolated from this plant is a likely new genus and species from the Chaetothryiales. Sida carpinifolia, which is in the Malvaceae family and has been found in Brazil and Argentina, has also been found to contain swainsonine, however, no fungal endophyte has been identified yet.   Deana Baucom presented her research on identification and characterization of polyketide synthases (PKS) from Undifilum oxytropis. Using sequence alignment tools and the as yet unassembled genome of the fungus, she discovered 22 PKSs that had homology to other fungi.  She found Type I PKS of high reducing, partially reducing, and low reducing types, NRPS types, and hybrid PKS-NRPS types.  One PKS had very high amino acid identity with the melanin PKS from Alternaria.  One NRPS had very high amino acid identity with a virulence factor found in Alternaria and other fungi.  A PKS-NRPS hybrid had good homology with several Metarhizium species, and is speculated to function in swainsonine biosynthesis.   Barb Keith presented the research from the Sterling laboratory at Monatana State University. Locoweeds have been reported in Montana since the early 1900s, and are predominantly Oxytropis spp. such as O. sericea.  Previous work had shown that swainsonine increased in plants with shortened photoperiod, i.e. fall and winter.  When transplanted into a common garden, Oxytropis has been long lived, despite the very cold weather found in Bozeman. In contrast, Astragalus mollissimus, which is not normally found in the state, grows as an annual and appears very susceptible to freeze damage.  There were no differences in the survival rate of Oxytropis plants that contained (+) or did not contain (-) the Undifilum endophyte.  There were also no differences between the E+ and E- plants in terms of transpiration, photosynthesis, or stomatal conductance, when testing flowering plants in June and July over a several year period.  Studies to determine if endophyte presence helped A. mollissimus survive stress (transplanting) and cold temperatures, showed mixed results, with overall non-significant changes in photosynthesis.  In 2014 and 2015, there were mixed results, but overall non-significant differences between E+ and E- plants in flowers/stem, pods/stem, and seeds/pod, and the overall germination, and germination rate.   Daniel Cook presented a research update of the recent work from his lab. He presented an extensive list of Astragalus species that were reported to contain swainsonine and his results when testing from both fresh and herbaria specimens.  Around nine of the species tested did not contain swainsonine; the discrepancy was attributed to incorrect identification of the plant or collecting the incorrect plant or to low sensitivity in previous testing methods.  A few plants had very low levels of swainsonine and a few had only 1 plant out of a large number that tested positive for swainsonine.  Swainsonine identification was correlated with the South American Clade G group of Astragalus sp. and with the A. allochrous group.  His lab has found swainsonine in many Ipomoea species, although the endophytes have not been characterized yet.  He cooperated on a study looking at the effect of elevated CO2 and endophyte presence on A. mollissimus growth and crude protein.  E- plants with elevated CO2 grew large than E+ plants, but crude proteins were lower in E- plants.  No difference was found in swainsonine with elevated CO2.   Chris Schardl discussed seed-transmitted endophytic fungi, primarily those from grasses, Ipomoea, and locoweeds.  The fungi are diverse ascomycetes and most are cryptic and nonpathogenic.   For grasses and locoweeds, the endophytes grow between cells. Grasses have endobiotic (Epichloe) endophytes and epibiotic (Atkinsonella and some Balansia) endophytes.  Some Ipomoea species can have epibiotic Periglandula spp. endophytes, and others have endophytes of the fungal order Chaetothyriales which may also be epibiotic. Locoweeds have endobiotic (Undifilum) endophytes. Epichloe has different interactions with seed pre and post pollination.  Before pollination, the mycelia goes through the placental pore into the ovule, but not into the embryo sac.  After pollination, the mycelia goes throughout the embryo sac. Epichloe infects the anthers but not the pollen grains. Periglandula grows on the outside of Ipomoea leaves.  It gets into the ovules of young plants and can be found associated with capsule walls, seed coats, and embryos of older plants. Periglandula can be found between tissue types associated with the surface of tissues, for example between leaf primordial.   Rebecca Creamer gave an overview of work completed by her former graduate student, Mohammad Alhawatema on Slafractonia leguminicola.  The fungus, previously known as Rhizoctonia leguminicola, was demonstrated to be morphologically different from Rhizoctonia sp.  It also has ITS sequence and gpd sequence that were closest to Pleiochaeta setosa, an ascomycete.  The genus was renamed to Slafractonia.  The polyketide synthase gene associated with melanin biosynthesis was identified based on homology with other ascomycetes and the gene sequenced.  The sequence was used for RNAi using the pSilent vector.  After introduction into fungal protoplasts, the resulting silenced colonies were light in color, moving to a light gray color, compared to the black wild type cultures.  The lighter colored colonies had less expression of the melanin gene and had reduced swainsonine concentration compared to the wild type colonies.

Publications

None for the time period covered

Impact Statements

Four fungi that produce swainsonine were sequenced, although the genome assembly has not been completed

Date of Annual Report: 01/04/2017

Report Information

Annual Meeting Dates: 11/14/2016 - 09/15/2016
Period the Report Covers: 10/01/2015 - 09/30/2016

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

Aziza Noor – Molecular Biology, New Mexico State University, anoor@nmsu.edu

Marwah Neyaz – Dept EPPWS, New Mexico State University, marwane@nmsu.edu

David Thompson – AES, New Mexico State University, dathomps@nmsu.edu

Brief Summary of Minutes

W1193 Locoweed Regional Project Annual Meeting

Locoweed and its fungal endophyte: impact, ecology, and management

November 15, 2016

Gerald Thomas Hall 297, NMSU

9:00-4:00

Welcome – David Thompson, Director Agricultural Experiment Station, NMSU

Background of locoweed and its fungal endophyte – Rebecca Creamer

Microscopy Analysis of locoweed endophytes and Chaetothriales – Aziza Noor

Molecular Identification of locoweeds – Marwa Neyaz

Updates from Montana Common Garden Study – Tracy Sterling and Barb Keith

Swainsonine genes and role of swainsonine in virulence of Metarhizium to insects – Chris Schardl

Comparison of PKS among swainsonine-producing fungi – Aziza Noor

Update of locoweed and endophyte research – Daniel Cook

Preliminary survey for seed-transmitted fungal endophytes in diverse plant species – Chris Schardl

Discussion

Accomplishments

The group was welcomed by the project coordinator, David Thompson, who is also the Agricultural Experiment Station Director at New Mexico State University.  He talked about how regional projects work and several potential roles for project participants. Rebecca Creamer gave an overview and background of locoweeds and their associated fungal endophytes and focused on the work of her laboratory on the fungal endophytes. Information was presented on a possible ecological advantage to the locoweed plants for harboring the fungus.  A survey of the fungal microbiome of Chinese locoweed plants showed that plants infected with the Alternaria section Undifilum endophyte had fewer foliar pathogens associated with the plants.  This suggests a possible mutualist role to suppress pathogens for the endophyte. Aziza Noor, a PhD student working under Rebecca Creamer, presented her research showing the characterization of A. U. oxytropis, A. U. cinereum, and A. U. fulvum in locoweed plants using confocal microscopy and scanning electron microscopy.  She found that all three fungi grow between cells, primarily in the pith of stems, without causing any obvious damage to the plant or recognition by the plant.  The fungi were demonstrated to grow by addition of tissue to the hyphal tips and the growth rate for each fungus was shown to be more rapid initially, and then slowing to limited growth by 20 days and no growth by 30 days. Marwa Neyaz, a MS student working under Rebecca Creamer, presented her initial research searching for primer sets to differentiate between species (and varieties) of locoweeds.  She identified chloroplast primer sets that amplified a selection containing indels that were useful in differentiating between several species of locoweeds. MONTANA report from Tracy Sterling and Barb Keith - The role of the fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus and Oxytropis sericea) plant growth parameters was measured in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT. These 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.  There is not an endophyte effect for plant survival although there is a species survival difference with fifty percent of O. sericea plants surviving 3-years, regardless of endophyte status and no A. mollissimus plants surviving beyond 2-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 showing a statistical higher rate of transpiration this year. This effect was not seen in the previous three summers.  For O. sericea, presence of the endophyte does not affect fecundity.  Fecundity analysis for A. mollissimus is ongoing. Data analysis was averaged across age of plant. We are currently investigating whether is there is an age-related endophyte effect for these parameters. A legacy study was initiated in the garden by establishing O. sericea seedlings to evaluate the effect of previous endophyte exposure on the physiological responses of plants with and without the endophyte to determine if epigenetics are playing a role in plant response to the endophyte. This goal was accomplished by collecting seeds from 20, 1-year-old plants in 2014 (10 E+ and 10 E-); from these, five seedlings from each were established in Fall 2015. 48 of 50 seedlings survived overwintering regardless of endophyte. Again, no difference was detected in gas exchange or fecundity between E+ and E- plants. Although twenty-one percent more E- plants set seeds after the first over-wintering. From seeds collected this summer from the legacy plants, a second generation of plants free from the fungal endophyte will be established. 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. Christopher Schardl presented on collaborative research that demonstrated the swainsonine biosynthetic pathway.  The genes were determined first from a Chaetothyriales fungus that produces swainsonine, then correlated with sequence from Alternaria Undifilum oxytropis and Metarhizium anisopliae.  A key enzyme in the biosynthetic pathway was knocked out from M. anisopliae which eliminated swainsonine production.  The knockout did not change in its insect pathogenicity. Aziza Noor presented a talk on her research to compare the ketide synthase among swainsonine-producing fungi.  She developed a primer set that provides good separation of the various endobionts that produce swainsonine. Daniel Cook presented information in regard to a swainsonine screen of several Astragalus and Oxytropis species.  In the first screen, several Astragalus and Oxytropis species presumed to contain swainsonine based upon field reports of poisoning or non-specific methods of detection such as thin layer chromatography and a jack bean α-mannosidase inhibition assay were investigated.  22 Astragalus species representing 93 taxa and 4 Oxytropis species representing 18 taxa were screened for swainsonine using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.  Swainsonine was detected in 48 Astragalus taxa representing 13 species and 5 Oxytropis taxa representing 4 species.  Forty of the fifty-three swainsonine-positive taxa had not been determined to contain swainsonine previously using liquid or gas chromatography coupled with mass spectrometry. In the second screen, 31 Astragalus species in the taxonomic sections Densifolii, Diphysi, Inflati, and Trichopodi previously not known to contain swainsonine. Furthermore, to broaden the scope further, 21 species within the 8 sections of the Pacific Piptolobi and the small flowered Piptolobi were screened for swainsonine.  Swainsonine was detected for the first time in 36 Astragalus taxa representing 29 species using liquid and gas chromatography coupled with mass spectrometry.  Several taxonomic sections were highly enriched in species that contain swainsonine while others were not.  The list of swainsonine-containing taxa identified through these screens will serve as a reference for risk assessment and diagnostic purposes. Daniel Cook also presented information in regard to how two different swainsonine-containing Astragalus species responded to elevated CO2 concentrations. Measurements of biomass, crude protein, water-soluble carbohydrates and swainsonine concentrations were measured in the two respective chemotypes (i.e., positive and negative for swainsonine) of each species at near present-day ambient and elevated CO2.  Ultimately, changes in CO2 and endophyte status will likely alter multiple physiological responses in toxic plants such as locoweed, however it is difficult to predict how these changes will impact plant herbivore interactions. Chris Schardl discussed preliminary results of a survey of seed-transmitted endophytic fungi, primarily including those from grasses and Ipomoea.  The study is a sequencing study of the ITS region and the fungi identified are diverse ascomycetes and some are known pathogens, while others appear to be nonpathogenic.  Fungi included Cladosporium and Fusarium species. The group discussed cooperative research projects and resources that could be shared among the group.  Daniel Cook, Chris Schardl and Rebecca Creamer will continue to collaborate on identification of the key genes in swainsonine biosynthesis. They will write a grant for a large cooperative project with Chinese collaborators further addressing the seed-transmitted endophytic fungi. The entire group met, discussed the current status of locoweeds, locoism and fungal endophytes. A subset of the group worked together on cooperative research.  Several papers will be written from the collaborative work.  The subset set priorities for collaborative research for the coming year.

Publications

Cook, D., Gardner, D. R., Lee, S. T., Pfister, J. A., Stonecipher, C. A., & Welsh, S. L. (2016). A swainsonine survey of North American Astragalus and Oxytropis taxa implicated as locoweeds. Toxicon, 118, 104-111. Pfister, J. A., Cook, D., Panter, K. E., Welch, K. D., & James, L. F. (2016). USDA-ARS Poisonous Plant Research Laboratory: History and Current Research on Western North American Rangelands. Rangelands, 38(5), 241-249. Cook, D., Gardner, D. R., Roper, J. M., Ransom, C. V., Pfister, J. A., & Panter, K. E. (2016). Fungicide treatment and clipping of Oxytropis sericea does not disrupt swainsonine concentrations. Toxicon, 122, 26-30.

Impact Statements

Rebecca Creamer worked with Daniel Cook on microscopy of the Chaetothyriales-producing fungus from Ipomoea

Date of Annual Report: 11/28/2017

Report Information

Annual Meeting Dates: 11/07/2017 - 11/07/2017
Period the Report Covers: 01/01/1970 - 01/01/1970

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
Aziza Noor – Molecular Biology, New Mexico State University, anoor@nmsu.edu
Marwa Neyaz – Dept EPPWS, New Mexico State University, marwane@nmsu.edu
Erik Lehnhoff – Dept, EPPWS, New Mexico State University, lehnhoff@nmsu.edu
Yongtao Yu – College of Agriculture, Ningxia University, yongtao@nmsu.edu
Ram Nadathur – Molecular Biology, New Mexico State University, januj88@nmsu.edu

Brief Summary of Minutes

Daniel Cook presented an overview and timeline of research on locoweeds and their associated fungal endophytes, as well as other swainsonine-containing plants and fungi. The swainsonine-containing plants are found in the western USA and China (Astragalus and Oxytropis spp.), Australia (Swainsona), worldwide (Ipomoea- Convulvulaceae), and South America (Sida – Malvaceae). Ipomoea also contain calystegine toxins, which the plant produced. Some Astragalus species in the western USA are toxic due to swainsonine and others due to accumulation of selenium or nitrotoxin. Swainsonine causes disease by inhibiting alpha mannosidase, causing a lysosomal storage disease, and mannosidase II, leading to altered glycoprotein synthesis. He updated the current research efforts with brief reports on publications in 2016 and 2017 including those by Klypina et al (Weed Science), Cook et al. (Chemistry and Biodiversity), (The Rangeland Journal), (Toxicon), (G3), and (Toxicon).

Yongtao Yu, visiting scientist, gave a background of locoweed research in China and presented and update on recent unpublished research. Locoweeds that have caused problems in China include Astragalus variabilis and A. strictus, and Oxytropis ochrocephala, O. kansuensis, O. glabra, and O. glacialis. Data was presented from Guodong Yang and Jianhua Wang showing that when feeding radiolabelled lysine to endophyte, the label goes to pipecolic acid and then to swainsonine. When endophyte was grown on liquid media without nitrogen, mycelia dry weight peaked at least a week before swainsonine levels peaked. Growth of Undifilum oxytropis and swainsonine production was studied in liquid media. A pH of greater than 5.5 was optimal for swainsonine production. Addition of lysine to the media increase swainsonine production, while addition of pipecolic acid inhibited swainsonine production. In recent work, mutants of U. oxytropis were made using various mutagens including UV and EMS. Although the mutants have not been extensively tested, several were very low swainsonine producers.

Aziza Noor, a PhD student working with Rebecca Creamer, discussed her work on the microscopy analysis of the Chaetothyriales fungus associated with Ipomoea carnea. Using confocal and fluorescence microscopy, she showed that the fungus was found on the outside of leaves and stems, but not under the surface of the epidermis. The fungus was visualized in a dense network around trichomes and stomates without penetrating the leaves or stems. The work strongly suggests that the fungus grows solely epiphytic with the plants.

Marwa Neyaz, a MS student working under Rebecca Creamer, presented her molecular characterization of locoweed species and varieties. She used three primer sets to variable regions with the chloroplast and ITS primers to differentiating among species and varieties of Astragalus mollissimus and Astragalus lentiginosus. Nontoxic varieties clustered with other nontoxic varieties or those with low levels of toxicity. Also varieties from the same region clustered together. The toolset developed can be helpful with identification of locoweeds and predictive relationships among the plants.

Tracey Sterling and Barb Keith presented a update of locoweed work at Montana State University. The role of the fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus and Oxytropis sericea) plant growth parameters was measured in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT. These 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. There is not an endophyte effect for plant survival although there is a species survival difference with fifty percent of O. sericea plants surviving 3-years, regardless of endophyte status and no A. mollissimus plants surviving beyond 2-years. No A. mollissimus plants were alive in the spring of 2017 and no new plants were established. 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 showing a statistical higher rate of transpiration, but only for one of the 5 years analyzed. For both A. mollissimus and O. sericea, presence of the endophyte does not affect fecundity. Data analysis was averaged across age of plant. We are currently investigating whether is there is an age-related endophyte effect for these parameters.

A legacy study was initiated in the garden by establishing O. sericea seedlings to evaluate the effect of previous endophyte exposure on the physiological responses of plants with and without the endophyte to determine if epigenetics are playing a role in plant response to the endophyte. Seeds were collected from 20, 1-year-old plants in 2014 (10 E+ and 10 E-) (Fig. 1); from these, five seedlings from each were established in Fall 2015. There is no difference in survival rates between E+ and E- plants after two winters. Similarly there was not a detectable difference in gas exchange or fecundity between E+ and E- plants after two winters. Twenty-one percent more E- plants set seeds after the first over-wintering; however this discrepancy was not seen after the second over-wintering with 88% and 86% E- and E+ plants, respectively, setting seed.

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, germinated in the greenhouse and transplanted to the garden during spring 2017 (Fig. 1). Again, there is no detectable difference in gas exchange between E+ and E- plants 2 months post transplanting. 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.

Aziza Noor presented a talk on her research comparing the nucleotide sequence of the ketide synthase important in swainsonine biosynthesis (KS-SWA) among swainsonine-producing fungi. She found that it worked well to separate phylogenetic groups of fungi from a particular host, Alternaria fulva from A. cinerea. It also separated out the endophytes from Swainsona. Interestingly it grouped A. bornmuelleri and A. gansuense together, which are both phytopathogens that produce very low levels of swainsonine. The groupings of fungi were similar to clades produced from ITS primers. The variability within KS-SWA protein sequence was very low, which could be expected from this coding region.

Rebecca Creamer presented a project from a student in the Bridge program on isolation of fungi loosely associated with Oxytropis sericea plants with and without endophyte grown in a common garden in Utah. A minimal surface sterilization of 30 sec was done prior to isolation. The resulting fungi were identified using ITS sequence. Results showed many Alternaria species associated with the leaves of E+ plants, with a predominance of A. alternata, a known phytopathogen, although no disease symptoms were seen. E+ stems yielded both A. oxytropis and A. alternata. These results suggest a possible mutualist role to suppress pathogens for the endophyte via niche colonization.

The group discussed cooperative research projects and resources that could be shared among the group. Daniel Cook and Rebecca Creamer will continue to collaborate on endophytes associated with Swainsona sp and with endophytes associated with the Astragalus pubentissimus/pardilinus group. Chris Schardl and Rebecca Creamer will cooperate on a prospective grant for a large cooperative project with Chinese collaborators further addressing seed-transmitted endophytic fungi.

Suggested topics for research were microscopic analysis of germinating seeds of Astragalus and Ipomoea, identification of an endophyte within Sida, looking at multiple stresses and fire on antagonistic interactions, comparing the microbiome of field-grown plants, and conducting a total mineral analysis of field-grown plants.

Minutes Attachment:

2017 Report of W1193 Locoweed Project.docx

Accomplishments

Publications

Publications:  No group publications.  However, publications on the topic by collaborations and by members during 2017 are listed below. Alhawatema, M., Sayed, Cook, D., Creamer, R. 2107. RNAi-mediated down regulation of a melanin polyketide synthase (pks 1) gene in the fungus Slafractonia leguminicola.  World J. Microbiol. Biotech. 33:179. Cook, D., Donzelli, B.G.G., Creamer, R., Baucom, D. L., Gardner, D.R., Pan, J., Moore, N., Krasnoff, S.B., Jaromczyk, J.W., Schardl, C.L. 2017. Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3:Genes, Genomes,Genetics 7:1791-1797. Cook, D., Gardner, D.R., Martinez, A., Robles, C.A., Pfister, J.A. 2017. Screening for swainsonine among south American Astragalus species. Toxicon 139: 54 Cook, D., Gardner, D.R., Welch, K.D., Allen, J.G.  2017. A survey of swainsonine content in Swainsona species. The Rangeland Journal 39:213-218. Lu, H., Quan, H., Zhou, Q., Ren, Zhenhui, Xue, R., Zhao, B., Creamer, R. 2017.  Endogenous fungi isolated from three locoweed species from rangeland in western China. Afr. J. Microbiol. Res. 11:155-170.

Impact Statements

Date of Annual Report: 11/28/2017

Report Information

Annual Meeting Dates: 11/07/2017 - 11/07/2017
Period the Report Covers: 10/01/2016 - 09/30/2017

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
Aziza Noor – Molecular Biology, New Mexico State University, anoor@nmsu.edu
Marwa Neyaz – Dept EPPWS, New Mexico State University, marwane@nmsu.edu
Erik Lehnhoff – Dept, EPPWS, New Mexico State University, lehnhoff@nmsu.edu
Yongtao Yu – College of Agriculture, Ningxia University, yongtao@nmsu.edu
Ram Nadathur – Molecular Biology, New Mexico State University, januj88@nmsu.edu

Brief Summary of Minutes

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.

Minutes Attachment:

2017 Report of W1193 Locoweed Project.docx

Accomplishments

Publications

Publications:  No group publications.  However, publications on the topic by collaborations and by members during 2017 are listed below: Alhawatema, M., Sayed, Cook, D., Creamer, R. 2107. RNAi-mediated down regulation of a melanin polyketide synthase (pks 1) gene in the fungus Slafractonia leguminicola.  World J. Microbiol. Biotech. 33:179. Cook, D., Donzelli, B.G.G., Creamer, R., Baucom, D. L., Gardner, D.R., Pan, J., Moore, N., Krasnoff, S.B., Jaromczyk, J.W., Schardl, C.L. 2017. Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3:Genes, Genomes,Genetics 7:1791-1797. Cook, D., Gardner, D.R., Martinez, A., Robles, C.A., Pfister, J.A. 2017. Screening for swainsonine among south American Astragalus species. Toxicon 139: 54 Cook, D., Gardner, D.R., Welch, K.D., Allen, J.G.  2017. A survey of swainsonine content in Swainsona species. The Rangeland Journal 39:213-218. Lu, H., Quan, H., Zhou, Q., Ren, Zhenhui, Xue, R., Zhao, B., Creamer, R. 2017.  Endogenous fungi isolated from three locoweed species from rangeland in western China. Afr. J. Microbiol. Res. 11:155-170.

Impact Statements

Date of Annual Report: 12/11/2018

Report Information

Annual Meeting Dates: 11/06/2018 - 11/06/2018
Period the Report Covers: 10/01/2017 - 09/30/2018

Participants

Daniel Cook – USDA/ARS Poisonous Plant Lab, Logan, UT Daniel.cook@usda.ars.gov.us
Barbara Keith – Dept Land Resources & Environmental Sci, Montana State University, bkeith@montana.edu
Christopher Schardl – University of Kentucky, chris.schardl@uky.edu
Tracy Sterling – Dept. Land Resources & Environmental Sci, Montana State University, tracy.sterling@montana.edu
Rebecca Creamer – Dept EPPWS, New Mexico State University, creamer@nmsu.edu
Sumanjari Das –Biology, New Mexico State University, sdas@nmsu.edu
Marwa Neyaz – Plant & Environmental Sci., New Mexico State University, marwane@nmsu.edu
Erik Lehnhoff – Dept, EPPWS, New Mexico State University, lehnhoff@nmsu.edu
Ram Nadathur – Molecular Biology, New Mexico State University, januj88@nmsu.edu
Christopher Davies –Assoc. AES Director, Utah State University, chris.davies@usu.edu

Brief Summary of Minutes

Chris Davies briefly discussed the Multistate project system. He reiterated the necessity of clear impact statements for the group. There was a brief discussion on the possibility of broadening the group to include other toxin producing plant endophytes that affect animals and adding international collaborators.

Daniel Cook presented a historical overview of research on locoweeds and their associated fungal endophytes, as well as other swainsonine-containing plants and fungi. Locoweds have been a problem since Cortez moved out of Mexico into what is now the western US. Locoweeds caused problems for settlers to the western US and the cattle herds that were moved westward. The swainsonine-containing plants are found in the western USA and China (Astragalus and Oxytropis spp., Fabaceae), Australia (Swainsona, Fabaceae), worldwide (Ipomoea- Convulvulaceae), and South America (Sida – Malvaceae). Ipomoea also contain calystegine toxins, which the plant produces. Some Astragalus species in the western USA are toxic due to swainsonine and others due to accumulation of selenium or nitrotoxin.

Swainsonine causes chronic disease by inhibiting alpha mannosidase, causing a lysosomal storage disease, and mannosidase II, leading to altered glycoprotein synthesis. The animals with the greatest sensitivity to swainsonine are horses and goats followed by sheep, then cows, then rats, mice, and deer, and finally chickens, which are much less susceptible to swainsonine poisoning.

Oxytropis behaves as a long-lived perennial, while Astragalus functions as an annual in cold locations and biennial in warmer locations. However, growth and survival of both plant genera are dependent on rainfall. The plants are cyclic, leading to cyclic livestock losses. Locoweeds are highly palatable to cattle, but the animals prefer green grasses, so losses are highly dependent on temperature and moisture conditions.

Tracey Sterling and Barb Keith presented a update of locoweed work at Montana State University. The role of the fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus and Oxytropis sericea) plant growth parameters was measured in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT. These 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. There is not an endophyte effect for plant survival although there is a species survival difference with fifty percent of O. sericea plants surviving 3-years, regardless of endophyte status and no A. mollissimus plants surviving beyond 2-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 showing a statistical higher rate of transpiration, but only for one of the 6 years analyzed. For both A. mollissimus and O. sericea, presence of the endophyte does not affect fecundity. Data analysis was averaged across age of plant. We are currently investigating whether there is an age-related endophyte effect for these parameters.

A legacy study was initiated in the garden by establishing O. sericea seedlings to evaluate the effect of previous endophyte exposure on the physiological responses of plants with and without the endophyte to determine if epigenetics are playing a role in plant response to the endophyte. Seeds were collected from 20, 1-year-old 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, germinated in the greenhouse and transplanted to the garden during spring 2017.

In the first generation of plants free from the fungal endophyte, E- plants have a higher survival rate than E+ plants after 3 winters (66% and 56% survival, respectively). Similarly, E- plants in the second generation of plants free from the fungal endophyte also have a higher survival rate than E+ plants of the same generation after one winter (70% and 52% survival, respectively). This was not seen in the parental population where E+ plants had a slightly higher rate of survival after one and three winters.

The Post Farm received above average precipitation during the spring on 2018. Gas exchange measurements for all three generation of plants were conducted twice during the summer of 2018; once in June during which time the Post Farm received 2.7 inches of precipitation and again in late July when precipitation measured just 0.24 inches. In June, plants were at the immature seed pod stage and were post-seed shatter in July. There is no detectable difference in gas exchange between E+ and E- plants when either well-watered or drought stressed.

Neither the first generation nor the second generation of plants released from the fungal endophyte (E-) showed a difference in fecundity to plants with the fungal endophyte (E+) of the same generation. In the parental population (plants established in 2013) E+ plants exhibited an increase in seed pods/stem over that of E- plants for four of the five years of measurements. This increase in seed pods/stem was not seen in E+ plants established in other years.

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.

Marwa Neyaz, a PhD student working under Rebecca Creamer, presented her proposal for molecular characterization/differentiation of four complicated Astragalus locoweed species, Astragalus wootoni, A. allochrous, A. pubentissimus, A. pardalinus. She showed that three primer sets to variable regions with the chloroplast and ITS primers did not distinguish between the species. The fungi isolated from the plants were not differentiated into distinct groups either. This suggests that there may be incomplete speciation within one or more of the problematic species.

Sumanjari Das, a Biology PhD student working with Rebecca Creamer, presented her research on the role of swnT in transporting swainsonine and slaframine out of Slafractonia leguminicola. She is also trying to determine the slaframine biosynthetic pathway in the fungus.

Ram Nadathur, a Molecular Biology PhD student working with Rebecca Creamer, presented his research looking at the regulation of swnK in Alternaria oxytropis and A. bornmuelleri. He found that there were lower transcript levels for swnK in the second fungus than the first, which correlates with their swainsonine production. He also presented his findings on three MAPKs from A. oxytropis and their relatedness with those from other Alternaria species.

Rebecca Creamer presented a small project on isolation of fungi and bacteria loosely associated with Oxytropis sericea plants with and without endophyte grown in a common garden in Utah. A minimal surface sterilization of 30 sec was done prior to isolation. The resulting fungi were identified using ITS sequence and the bacteria through 16S sequence. Results showed many fungi were associated with the leaves and stems of E+ plants, with a predominance of A. alternata, a known phytopathogen. The E- plants (leaves and stems) yielded high numbers of bacteria, which were primarily known epiphytes. These results suggest a possible mutualist role to suppress bacteria for the endophyte via niche colonization.

The group discussed cooperative research projects and resources that could be shared among the group. Daniel Cook and Rebecca Creamer will continue to collaborate on the Astragalus pubentissimus/pardilinus group, and the Slafractonia swnT and slaframine project.

Suggested topics for research were: looking at castanospermine for its relatedness to swainsnonine, testing antisense production of swainsonine, adding inhibitors of plant/fungal interactions, determining how the fungus/plant change across the landscape and how affects those changes, determining what swainsonine does to the fungus, determining the mechanism by which endophytes are lost from Oxytropis, but not Astragalus at a specific geographic location.

Accomplishments

Publications

Publications:  No group publications.  However, publications on the topic by collaborations and by members during 2018 are listed below. Noor, A.I., Nava, A., Cooke, P. Cook, D. Creamer, R. 2018. Evidence of non-pathogenic relationship of Alternaria section Undifilum endophytes within three host locoweed plant species.  Botany 96:187-200. Harrison, J.G., Parchman, T.L., Cook, D., Gardner, D.R., Forister, M.L. 2018. A heritable symbiont and host-associated factors shape fungal endophyte communities across spatial scales. Journal of Ecology. 1-13. <a href="https://doi.org/10.1111/1365-2745.12967">https://doi.org/10.1111/1365-2745.12967</a>. Cook, D., Gardner, D.R., Martinez, A., Robles, C., Pfister, J.A. 2018. A screen for swainsonine among South American Astragalus species. Toxicon. 139:54-57. <a href="https://doi.org/10.1016/j.toxicon.2017.09.014">https://doi.org/10.1016/j.toxicon.2017.09.014</a>.

Impact Statements

Research based facts of the locoweed-fungal endophyte system have been disseminated throughout several states

Date of Annual Report: 12/11/2019

Report Information

Annual Meeting Dates: 10/29/2019 - 10/29/2019
Period the Report Covers: 10/01/2018 - 09/30/2019

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, New Mexico State University, sdas@nmsu.edu
Marwa Neyaz – Plant and Environme, New Mexico State University, marwane@nmsu.edu
Erik Lehnhoff – Dept, EPPWS, New Mexico State University, lehnhoff@nmsu.edu
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, New Mexico State University, jturner@nmsu.edu

Brief Summary of Minutes

Chris Davies briefly discussed the Multistate project system. He explained that the project would need to be renewed in 2020 and clarified that the timeline required a January 15 deadline for the proposal so that the project could be renewed by the end of Sept 2020. He made the suggestion to retain the research project instead of applying for a WERA-type project. There was a brief discussion and agreement among the group to broaden the group to include not only other toxin producing plant endophytes that affect animals, but also to poisonous plants in general to increase the number of participants. This will entail recruiting much more widely for help in writing the proposal, and devising a list of 10 peer reviewers to go along with the proposal.

Rebecca Creamer presented an overview of research on locoweeds and their associated fungal endophytes, as well as other swainsonine-containing plants and fungi. Swainsonine causes the chronic disease locoism by inhibiting alpha mannosidase and mannosidase II. There was a discussion of the swainsonine biosynthetic pathway and the diversity among the PKS, NRPS, and hybrid NRPS/PKS found within the locoweed endophyte, suggesting a novel origin for some of these secondary metabolites. There was an update on the collaborative project with Montana State on isolation of fungi and bacteria loosely associated with Oxytropis sericea plants with and without endophyte grown in a common garden. A minimal surface sterilization of 1 min in bleach was compared with a 5 min treatment in bleach. The resulting fungi were identified using ITS sequence and the bacteria through 16S sequence. Results showed many fungi were associated with the leaves and stems of E+ plants, with a predominance of A. alternata, in leaves (with 1 min bleach) and A. oxytropis in stems (5 min bleach). The E- plants with 1 min bleach treatment (leaves and stems) yielded high numbers of gram positive bacteria, which were primarily Bacillus sp. These results suggest a possible mutualist role to suppress bacteria for the endophyte.

Marwa Neyaz, a PhD student working under Rebecca Creamer, presented her research on molecular comparison of the SWN cluster across all fungi identified to contain swnK. The fungi separated by order based on which of the SWN genes they contained. Most Pleosporales lacked several of the genes, while most Onygenales and Hypocreales contained all of the genes in the SWN cluster.

Ram Nadathur, a Molecular Biology PhD student working with Rebecca Creamer, presented his findings on three MAPKs from A. oxytropis and their relatedness with those from other Alternaria species, with the concept that these could help with pathogenicity and perhaps seed infection and survival.

Tracey Sterling presented a update of locoweed work at Montana State University. The role of the fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus and Oxytropis sericea) plant growth parameters was measured in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT. These 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. There is not an endophyte effect for plant survival although there is a species survival difference with fifty percent of O. sericea plants surviving 3-years, regardless of endophyte status and no A. mollissimus plants surviving beyond 2-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 showing a statistical higher rate of transpiration, but only for one of the 6 years analyzed. For both A. mollissimus and O. sericea, presence of the endophyte does not affect fecundity. Data analysis was averaged across age of plant. We are currently investigating whether is there is an age-related endophyte effect for these parameters.

A legacy study was initiated in the garden by establishing O. sericea seedlings to evaluate the effect of previous endophyte exposure on the physiological responses of plants with and without the endophyte to determine if epigenetics are playing a role in plant response to the endophyte. Seeds were collected from 20, 1-year-old 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, germinated in the greenhouse and transplanted to the garden during spring 2017.

In the first generation of plants free from the fungal endophyte, E- plants have a higher survival rate than E+ plants after four winters (52% and 38% survival, respectively). Similarly, E- plants in the second generation of plants free from the fungal endophyte also have a slightly higher survival rate than E+ plants of the same generation after two winter (38% and 34% survival, respectively). This was not seen in the parental population where after two winters the survival rate was the same for E- and E+ plants and E+ plants had a higher survival after four winters (33% E- and 46% E+ survival). However, the E+ and E- parental population plants had a similar survival rate of 20% after 6 winters.

The Post Farm received above average precipitation during the spring and summer of 2019. Gas exchange measurements for all three generation of plants were conducted twice during the summer of 2019; once in June during which time the Post Farm received 2.2 inches of precipitation and again in August when precipitation measured just 0.39 inches, although precipitation for July (2.6 inches) was well above average. In June, plants were at the immature seed pod stage and were post-seed shatter in August. As also report for 2018, there is no detectable difference in gas exchange between E+ and E- plants when either well-watered or drought stressed.

E- plants in both the parental population and the 1^st generation of plants released from the fungal endophyte showed an increase in the number of seeds/pod in 2019 as compared with E+ plants for the same generation. This increase was not seen in the 2^nd generation plants nor in any other year for each population. The parental population (plants established in 2013) E+ plants exhibited an increase in seed pods/stem over that of E- plants for four of the six years of measurements. The increase in seed pods/stem in E+ plants is not seen in subsequent generation. The presence (E+) or absence (E-) of the fungal endophyte does not influence germination of seeds collected from any of the generations over the years. Future studies will investigate if the presence of the fungal endophyte affects seed viability. Volatile compounds from 1^st generation E+ and E- plants were collected from the field plants during June when plants were flowering and again in August after seed shatter. As expected, there was a significant difference in the amount of volatiles released by plants in June as compared to August. There was not a significant difference in the volatiles being released from E+ plants and E- plants with the exception of an unknown sesquiterpene which was significantly up in E- plants in August as compared to the E+ plants. However, several terpenes were slightly and consistently elevated in E- plants compared to E+ plants both in June and August. Work is continuing to identify these terpenes and volatile collections will be repeat during the spring and summer of 2020.

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.

There was a general discussion of a subset of the group cooperating on a Western SARE grant application. Jason Turner will help identify interested ranchers. Tracy Sterling will contact an extension agent in Montana. Erik Lehnhoff suggested a project to develop a predictive model for likely problems with Astragalus and Oxytropis plants based on weather parameters. There were suggestions for obtaining historical data about severe locoweed years/locations, since the grant period is not long enough to record many outbreaks.

Accomplishments

The entire group met, discussed the current status of locoweeds, locoism and fungal endophytes. A subset of the group worked together on cooperative research. Several papers will be written from the collaborative work. The subset set priorities for collaborative research and grants for the coming year. Rebecca Creamer’s former student worked with Daniel Cook on work to compare the swnK from different swainsonine-producing fungi. That work will be submitted for publication soon. She also worked with Daniel Cook to finish up work using microscopy to characterize the association of the Chaetothyriales fungus associated with Ipomoea sp. One of Rebecca Creamer’s students collaborated with Daniel Cook on a phylogenetic comparison of Astragalus mollissimus and A. lentiginosus varieties that has been submitted for publication. The same student worked with Daniel Cook on characterization of locoweeds and their fungal endophytes from Argentina. Tracy Sterling and her group at Montana State University collaborated with an undergraduate student at New Mexico State University working with Rebecca Creamer studying the bacteria and fungi associated with locoweeds growing in a common garden in Montana. Student training: <ul> <li>A graduate student from Argentina worked on locoweeds with Daniel Cook at the ARS, Poisonous Plant Research Laboratory in Logan, Utah and also interacted with Rebecca Creamer.</li> <li>Three international PhD students currently working with Rebecca Creamer at NMSU in collaboration with Daniel Cook.</li> <li>Two underrepresented minority students (Native American) working with Rebecca Creamer, one in collaboration with Tracy Sterling.</li> </ul> International collaborations: <ul> <li>Collaborations have been established with three researchers at different universities in China on locoweeds and their fungal endophytes.</li> </ul>

Publications

No group publications.  However, publications on the topic by collaborations and by members during 2019 are listed below. Martinez, A., Robles, C., Roper, J.M., Gardner, D.R., Neyaz, M., Joelson, N., Cook, D. 2019. Detection of swainsonine-producing endophytes in Patagonian Astragalus species. Toxicon. 117:1-6. <a href="https://doi.org/10.1016/j.toxicon.2019.09.020">https://doi.org/10.1016/j.toxicon.2019.09.020</a>. Cook, D., Lee, S.T., Panaccione, D.G., Leadmon, C.E., Clay, K., Gardner, D.R. 2019. Biodiversity of Convolvulaceous species that contain ergot alkaloids, indole diterpene alkaloids, and swainsonine. Biochemical Systematics and Ecology. 86. <a href="https://doi.org/10.1016/j.bse.2019.103921">https://doi.org/10.1016/j.bse.2019.103921</a>.

Impact Statements

W1193 has resulted in dissemination of research-based facts of the locoweed-fungal endophyte system throughout several states.