Brief Summary of Minutes of Annual Meeting

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

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.

None for the time period covered