Integrating thistle rust into weed management of Canada thistle

Research Objective 1: Assess how thistle rust responds to conventional and reduced tillage practices.

An integration of thistle rust as a Canada thistle biocontrol in agricultural systems requires a clear understanding of the pathogen's response to management tactics. Thistle rust spores remain dormant within Canada thistle debris on the soil surface until they come in contact with a new host. Soil tillage has the potential to bury, transport, or destroy thistle rust spores which are responsible for transmission of the pathogen. This objective is designed to examine the potential for tillage to impact the spread and establishment of the thistle rust pathogen.

Cooperator Farm

During the summer of 2020, producer Ole Norgaard cooperated in the establishment of a field study on his organically certified farm near Shonkin, Montana. The farm has pre-existing Canada thistle infestations which contain varying degrees of naturally established thistle rust infection. The preliminary study explored tillage impacts on thistle rust establishment and spread. At Mr. Norgaard's farm, four discrete Canada thistle patches were identified, flagged and GPS mapped. Two of the patches contained varying degrees of symptomatic rust infected Canada thistle, while the other two patches showed no signs of infection. Tillage occurred in one patch containing thistle rust infection and one patch with no signs of infection, using the cooperator’s tillage method of choice. The remaining two patches were left untilled throughout the rest of the growing season. Prior to tillage, vegetation growth data was collected along 20-meter transects, placed through the center of each thistle patch. Ten 1-m² sample frames were placed at even intervals along the transects. Vegetation density data was collected for symptomatically infected thistle stems and total thistle stems within each frame.  Additionally, percent vegetation cover was estimated within frames, including % symptomatically infected Canada thistle, % asymptomatic Canada thistle, % crop, % other, and % bare ground.

Mr. Norgaard decided to plant alfalfa as a perennial forage crop in the fall of 2020. During the 2021 growing season, sampling methods were repeated in within the same four discrete thistle patches that were monitored in 2020. Mr. Norgaard did not till the soil or swathe the field because the alfalfa crop was in the early to mid-vegetative stage of growth. Low moisture conditions in the summer of 2021 led to poor establishment of Mr. Norgaard's alfalfa crop, and an expansion of the Canada thistle populations within the fields. As a result, Mr. Norgaard decided to remove the fields from organic certification in the fall of 2021. The fields which hosted this study were sprayed with 123.5 oz./hectare of glyphosate in the fall, followed by seeding of a forage mixture. 

As a response to the new management, the methods for the 2022 sampling period were altered to best accommodate the project objectives. The new objectives at the Norgaard Farm focused on re-evaluation of the original four thistle patches within the crop fields, with a new evaluation of thistle patches in a grazed pasture that borders Mr. Norgaard's crop fields. The goal was to determine if there were any differences in thistle density and the density of thistle stems with rust symptoms between the two habitat types at the Norgaard Farm. The crop fields were revisited in June 2022 where all four experimental thistle patches were sampled under previously established protocols. Three additional thistle patches, located within grazed pastures along Mr. Norgaard's crop fields, were also sampled using the original protocols. Canada thistle stem density and density of thistle stems with rust symptoms data were analyzed in RStudio using a generalized linear model with the gaussian distribution. Comparisons were made between thistle growth and rust symptoms from 2022 in the crop field habitat and the pasture habitat, and analysis was conducted in RStudio. The margin of each thistle patch was also mapped with an Emlid Reach RS2 GNSS receiver on June of 2022, and point vectors were taken at the location of all observations of symptomatically infected Canada thistle within each patch. The data was analyzed in QGIS on the WGS 84 Pseudo-Mercator coordinate reference system. Patch areas from each patch polygon was derived in QGIS, and RStudio was used to evaluate the difference in mean patch areas between the crop field habitat and the pasture habitat. 

Montana State University (MSU) Fort Ellis Research Farm

Integration of thistle rust into tillage is being evaluated at the MSU Ft. Ellis Research Farm in Bozeman, Montana. The research fields at the Ft. Ellis research farm are approximately 0.34 acres (0.14 hectares) each, with a mean annual precipitation of 56 cm and a mean annual air temperature of 6°C (USDA NRCS, 2019).

During the summers of 2020 and 2021, discrete thistle patches with pre-existing rust infections were surveyed and mapped within four organically certified fields. Each field was uniformly seeded with an organic green manure barley crop during the spring of 2020, at a rate of 197-lbs/hectare. In July, prior to Canada thistle anthesis, a census of symptomatically infected Canada thistle was taken from each thistle patch in all four replications. Additionally, asymptomatic Canada thistle stem density was counted within 1-m² sample frames per thistle patch, using a simple random sampling method. After thistle assessment, all four fields were randomly split into a reduced tillage mowing termination and a conventional tillage disc termination, with nearly equal representation of thistle rust in each treatment. The mowing termination was performed with a 72” wide flail mower set to leave 4” stubble. Disc cultivation was set at an 8” soil depth, and three passes were made per conventional tillage treatment. In the fall of 2020, a green manure mixture of organic winter peas and winter triticale was seeded into all four fields at a rate of 81-lbs/acre winter pea and 75-lbs/acre winter triticale. Sampling protocols were repeated in July of the following summer, prior to Canada thistle anthesis. Reduced and conventional tillage events were conducted within assigned treatment areas immediately after sampling, followed by a secondary tillage treatment that took place in September. In May of 2022, the fields were prepared for seeding with respective reduced and conventional tillage treatments. Each field was sown with spring wheat seed, immediately after seedbed preparation. The spring wheat crop will be grown to full maturity, and the final sampling event will take place prior to harvest. All sampling protocols will be repeated, with the addition of a final biomass and crop yield assessment.

Canada thistle stem density and the density of thistle rust infected Canada thistle stems was evaluated in RStudio. Data was analyzed between the years 2020 and 2021, and between the disc and mowed treatments. A Levene test was used to check the assumptions of equal variance in the data. As a response to the Levene tests, Welch two sample t-tests were used to compare the effects of tillage and years on thistle stem density and density of rust infected thistle stems. 

Research Objective 2: Compare the impacts of cropping management between rust-infected Canada thistle and non-infected Canada thistle. 

The experiments under this objective consist of a greenhouse study and a field study that assess the effects of thistle rust on Canada thistle competitiveness and spread. The greenhouse study is designed to look directly at the growth responses of thistle rust inoculated Canada thistle when it is under stress from crop competition, and within a controlled environment. The field study is designed to evaluate the performance of the biocontrol agent in a setting that more closely represents a "real world" cropping system.

Montana State University Greenhouse

The effects of crop competition on non-inoculated (control) and thistle rust inoculated Canada thistle was evaluated at the MSU greenhouse in Bozeman, MT.  Ten-inch diameter, eight-inch-deep greenhouse pots were sown with 1-gram (±0.2-grams) cuttings of adventitious Canada thistle rhizome, sourced from wild thistle populations in Gallatin County and Hill County Montana. Once the rhizome produced basal thistle rosettes, the pots were randomly assigned to thistle rust inoculated or control treatments. Treatments were segregated into separate greenhouses to prevent unintentional rust infection in the non-inoculation treatment. Each pot in the inoculated treatment received five-mL thistle rust spores topically applied to Canada thistle rosettes. The spores were sourced from symptomatically infected thistle stems in Gallatin County, and prepared by grinding symptomatic thistle leaves into a dry powder. Inoculations occurred at an average temperature of 22°C, and were misted once a day with deionized water for 72 hours after inoculation. Temperature and water applications were held constant for the duration of the study and supplemental lighting was used during the winter months. Nutrients were added bi-weekly in the form of a water-soluble 20-20-20 NPK fertilizer. Individual greenhouse pots were randomly rotated bi-weekly to prevent microclimate impacts. A competition treatment was nested into the control and thistle rust inoculated pots to simulate the impacts of a multi-phase crop rotation on inoculated thistle. The levels of competition included Canada thistle grown by itself (monoculture), Canada thistle grown with a crop, and a crop grown by itself (see figure 1).

A 4-phase crop rotation was used to assess the performance of thistle rust inoculated Canada thistle in response to competition from a multi-phase crop rotation consisting of phase 1.) fallow with 1-gram (±0.2-grams) of Canada thistle rhizome per pot, phase 2.) spring wheat seeded at 100 kg/hectare, phase 3.) forage peas seeded at 89 kg/hectare and, phase 4.) safflower seeded at 33 kg/hectare. Canada thistle was grown within its original pot throughout the four phases. Harvests occurred at the crop maturity stage within each phase (approximately every 3 months), where all above-ground thistle and crop vegetation were cut at soil-level and dried for 72 hours at 40.5°C, and weighed. After each harvest, the next crop phase was seeded at recommended seeding rates, and thistle was allowed to regrow. Once Canada thistle rosettes re-established, the inoculated treatment received a repeated application of thistle rust spores following the original protocols. Each trial was terminated after harvest of phase 4. At the termination of each trial the thistle rhizome was removed from pots, cleaned of soil and debris, dried, and weighed.

A relative competition index was used to evaluate the performance of above-ground Canada thistle growth among treatments. The thistle monoculture level of competition acted as an index for maximum potential growth, where Canada thistle had no interspecific competition to impact its growth. In contrast, the polyculture level represented a system of competition, where Canada thistle development was directly influenced by the growth of crop species throughout the 4-phase rotation. The relative competition index is a measure of plant performance which contrasts monoculture with polyculture dry biomass where P_monoculture is the average Canada thistle performance (biomass) in monoculture and P_polyculture is the average thistle performance (biomass) in polyculture.

RCI = ((P_{monoculture -} P_polyculture) ÷ (P_monoculture)) x 100%

Relative competition from above-ground biomass of Canada thistle within three competitive phases of the crop rotation in trials one and two was calculated in RStudio. A linear mixed effects model was fit with relative competition as the response to inoculation treatments (inoculated & control), rotational phase (wheat, pea, safflower), and trial (one & two) as covariates.

Rhizome performance was compared for both treatments using a relative growth rate (RGR) that calculated the difference of rhizome weight between the beginning and end of each trial. The pots containing Canada thistle were all initially sown with 1-gram (±0.2-grams) pieces of rhizome. Relative growth of Canada thistle rhizome within the control, inoculated, monoculture, and polyculture treatments were evaluated after the four phases of growth. In the following RGR equation W₁ represents initial rhizome weight at planting, W₂ represents dry rhizome weight after 4 phases of growth, and t is the total days of growth for each trial.

  RRG_treatment = (lnW₂ -lnW₁) ÷(t)

The relative growth rate of Canada thistle rhizome (grams/day), grown in monoculture and polyculture within trials one and two, was evaluated in RStudio. Linear mixed effects model was fit with relative growth rate as the response to inoculation treatments (inoculated & control), levels of competition (monoculture and polyculture), and trial (one & two) as covariates.

Montana State University Post Agronomy Farm:

The impacts of the thistle rust biocontrol on Canada thistle growth is being evaluated at the MSU Post Agronomy Farm in Bozeman, Montana. The research fields at the site are each 0.02 hectares with a mean annual precipitation of 48 cm and a mean annual temperature of 7°C (USDA NRCS, 2019). This experiment combines biological, mechanical, and cultural controls for Canada thistle infestations within agroecosystems. During the spring of 2020, 1-gram cuttings of thistle rhizome were planted in two separate fallow fields. Soil samples were taken from each field for nutrient comparisons, and Delmhorst model GB-1 gypsum blocks were installed 15 centimeters below the soil surface for soil moisture monitoring. Each field contains 10 thistle rust inoculated and 10 non-inoculated Canada thistle. To reduce potential for unintentional thistle rust inoculation of the non-inoculated control treatment, the controls were intentionally planted in the predominant upwind direction, 12 meters away from the inoculated treatments. During the summer of 2020, both fields were maintained in fallow so that the planted rhizome could establish. Thistle growth data was collected in the fall within 1-m² sample frames centered over the original rhizome planting locations. Data collection included thistle stem density, density of thistle stems showing rust symptoms, % cover asymptomatic thistle, % cover symptomatic thistle, % cover crop, % cover bare ground, and % cover other. Fall inoculations were performed during an optimal climate range, where temperatures were forecasted between 8 and 25°C. A maximum of five thistle rosettes were inoculated within 1-m² sample frames centered over the original rhizome planting locations. Each inoculation consisted of a 5-mL application of thistle rust spores, applied directly to thistle rosettes. The spores were sourced from symptomatically infected thistle stems in Gallatin County, and prepared by grinding symptomatic thistle leaves into a dry powder. Thistle was allowed to senesce naturally in each field, which were left no-till to avoid disturbance of patch development. 

In the spring of 2021, 80-lbs/acre of forage barley was drill seeded into each no-till field with an in-furrow application of 13-36-0 fertilizer at a rate of 100-lbs/acre. Thistle was allowed to freely grow through the summer within the barley crop. Data collection occurred at the mealy ripening stage of the barley kernels. An Emlid Reach RS2 GNSS receiver was used to map the spread of thistle patch boundaries, and collect total patch area data for each treatment. Sampling of three additional transects was conducted, west and east from the original planting locations, to account for the spread of thistle beyond the original planting locations. Each transect was 20-m long, with ten 1-m² samples taken at every 0.5-m interval. Data collection followed the same protocols that were established in 2020. Each field was swathed with a gas-powered trimmer, hand raked, and cleared of above-ground biomass following data collection. The fields were left no-till, and thistle rhizomes were allowed to re-establish through the fall. Once fall climate conditions reached an optimal range, a maximum of five thistle rosettes were inoculated within all 1-m² sample frames along the four transects in the inoculated treatment. Inoculations were only applied within sample frames containing live thistle rosettes, following protocols established in 2020. 

Analysis was conducted in RStudio, comparing thistle stem density between the inoculated and non-inoculated treatments over 2020 and 2021. A linear mixed effects model was fit with thistle stem density as a response to treatment levels (inoculated & non-inoculated), levels of growing season (2020 & 2021), and levels of block (A & B) as covariates. The spatial spread of the planted Canada thistle at the Post Agronomy Farm was derived in Arc GIS as total patch areas (m²), and analyzed in RStudio using a generalized linear model.

In the spring of 2022, spring wheat was drill seeded in the two no-till fields at a rate of 85-lbs/acre. The sampling protocols that were established in 2021 will be repeated at the harvest-ready stage of wheat in 2022. Above ground thistle biomass, crop biomass, and grain yield will be collected from all 1-m² samples as a conclusion to the study.