Brief Summary of Minutes of Annual Meeting

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.

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.

Rebecca Creamer, and her students worked with Daniel Cook on microscopy of in vivo and in vitro growth of Alternaria oxytropis, A. fulva, and A. cinerea that was published. They also worked on Chaetothyriales-producing fungus from Ipomoea carnea.  That work is written for publication. They also collaborated on work to compare the swnK from different swainsonine-producing fungi.  That work is being written for publication.  Rebecca Creamer’s student also collaborated with Daniel Cook on a phylogenetic comparison of Astragalus mollissimus and A. lentiginosus varieties.  That work is being written for publication.

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. https://doi.org/10.1111/1365-2745.12967.

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. https://doi.org/10.1016/j.toxicon.2017.09.014.