Brief Summary of Minutes of Annual Meeting

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.

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.

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.

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:

• 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.
• Three international PhD students currently working with Rebecca Creamer at NMSU in collaboration with Daniel Cook.
• Two underrepresented minority students (Native American) working with Rebecca Creamer, one in collaboration with Tracy Sterling.

International collaborations:

• Collaborations have been established with three researchers at different universities in China on locoweeds and their fungal endophytes.

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. https://doi.org/10.1016/j.toxicon.2019.09.020.

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. https://doi.org/10.1016/j.bse.2019.103921.