Brief Summary of Minutes of Annual Meeting

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

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.

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.