Glyphosate resistance in wild parsnip

Tyler Baxby

0793160

AGR4460

December 12^th, 2019

University of Guelph

Glyphosate resistance in wild parsnip

A literature review presented by Tyler Baxby

For AGR-4460: Research project II

To: Professor Francois Tardif

Date: December 12^th, 2019

1. Introduction:

Recognizing how human activity changes biodiversity is becoming increasingly important, more than ever (Potts et al. 2010). Bill 64 is a legislated provincial act banning the use of Cosmetic Pesticides across the Province of Ontario. It was created with the prospect of superseding all the municipal by-laws, into one easily understandable document. However, the creation of the policy has been found to be based off of the same misinformation which had been used in the creation of municipal by-laws across Ontario. I have witnessed many municipalities (including my own city of Kingston, Ontario) proceed through the process of banning the use of horticultural (lawn care) pesticides within their municipal regions; these processes have been based solely on the basis of public opinion where they are dangerous for both the environment and humans alike. Bill 64 was passed through legislation, there are various abundant populations of wild parsnip emerging. There are potential dangers toward humans with these developments.

The spread of invasive species within ecosystems is a primary driver of change. Many invasives reach such levels they negatively influence biodiversity. (Rejmánek et al.2005). Wild parsnip (Pastinaca sativa) is a noxious perennial weed, that is a member of the carrot (Apiaceae) family. It is an invasive and also a public health hazard. As an invasive, it has a tendency to displace natural vegetation. This occurs especially on roadsides. In addition, this plant contains furanocoumarins in its sap. These molecules, when into contact AGR4460 with the skin, make it sensitive to UV rays, causing a condition called phytophotodermatitis. (Bowers,1999; Rietschel, et al. 1995).

This is potentially dangerous for outside workers as well as members of the public. (Zangerl and Berenbaum 1987). While roadside populations of wild parsnip were historically controlled with herbicides such as 2,4-D, the introduction of the cosmetic pesticides Ban Act (Bill 64) in 2008 has caused many jurisdictions to adopt alternative measures. Some have relied only on mechanical control while others have been using glyphosate extensively. I and others in the industry have observed in the last few years that glyphosate appears to be less effective at controlling some wild parsnip populations. One hypothesis is that these populations may have developed resistance to this herbicide. The purpose of this project is therefore to test that hypothesis.

• Proposed Methods

This project will involve a comprehensive review of the available existing research on known potential risks and the associated costs of wild parsnip as a preliminary study.

This will focus on the potential ramifications of glyphosate weed control usage. This literature review will outline information on the class 9 herbicide glyphosate. Class 9 pesticides are defined as a chemical that cannot be purchased for cosmetic use unless there is an exception (https://www.ontario.ca/page/classification-pesticides).

This review will also summarize published research regarding wild parsnip and what is already known about its management. Therefore, the additional part of this project outlines any potential drawbacks of excessive herbicide usage and indicate possible resistance of glyphosate and associated impacts on the environment.

• Local distribution of wild parsnip:

These invasives spreads quickly and has readily adapted to grow within these environments like abandoned yards, meadows, open fields, roadsides and railway embankments (Cain et al. 2010). In Canada, scattered populations of wild parsnip have been reported in every province and territory except Nunavut (NCC 2019). Most recently, in South Eastern and South Western Ontario, invasives have aggrandized and become more abundant. This is a problem because of the rapid dispersion and the abundant spread of invasive plants over the last two decades, have influenced natural ecosystems by changing the diversity to suit the invasive plant (Tassie and Kellie 2014).

• Noxious Weeds in Ontario:

2.1 Description of Wild Parsnip

Noxious is defined as physically harmful or destructive to living beings (Merriam-Webster. 2019.) Wild parsnip is not an exception as it has developed as a noxious weed in Ontario. It is classified as a towering, monocarpic perennial, that is also known as poison parsnip, common parsnip, bird’s nest, and hart’s eye (Tassie and Kellie. 2014.).

Wild parsnip is native to Eurasia but was introduced to North America shortly after European settlement, as an important crop for cultivation and food security (Cain et al. 2010; Darbyshire, 2003; Tassie and Kellie. 2014.). Wild parsnip once brought over during settlement evaded cultivation and naturalized as a less edible wild type. As it escaped cultivation, it has become an invasive which is spreading across North America (Canada excluding Nunavut, and the eastern United States). (Tassie, D. and S. Kellie. 2014; Averill and DiTommaso 2007).

Wild parsnip is rapidly spreading across the continent from east to west and is considered a pernicious weed in most Canadian provinces and many states within the United States (Figures:1.a.b.c; Averill and DiTommaso, 2007; Jogesh, et al.2015). Wild parsnip typically develops an occasional, thin rosette of leaves within the first year of growth, the second year being commonly characterised by yellow flowers on a stalk capable of reaching 1.5 meters tall.

As it reproduces entirely by flat, spherical seeds, effective dispersal is achieved by wind and water, or via vectors such as, animals and agricultural equipment (Tassie, and Kellie. 2014; Cain et al, 2010). Wild parsnip needs generally two years to mature, at which time, it flowers and then dies.

• Toxicity of wild parsnip

Wild parsnip is known to produce a sap that contains furanocoumarins – a class of chemical compound; which can cause a variety of negative health implications to human and livestock. (Baskin and Baskin 1979; Zangerl 1990).

Furanocoumarins are secondary metabolites produced in foliage and seeds of wild parsnip and other species. (Herms & Mattson,1992). While these compounds are abundant in wild parsnip, they have been selected out of cultivated varieties of parsnip (Cain et al., 2010). In addition, cultivated parsnip is distinguished from its wild counterpart by having a larger edible taproot. Wild parsnip contains a high concentration of a subclass of furanocoumarins called psoralens (Bowers,1999). Psoralens cause skin photosensitisations through interactions with DNA and induction of programmed cell death (Scott et al.,1976).

3. Phytophotodermatitis  

3.1 Human Health

Phytophotodermatis (PPD) is a skin condition caused by the interaction between sunlight radiation, human skin and specific plants (Finkelstein, et. al 1994). The initial contact causes hyperemia or increase blood flow causing redness. The redness induced by sunlight radiation can develop into blisters and attritions/scrapes (Rietschel, et al.,1995; Bowers,1999). The electromagnetic wavelengths responsible for PPD range between 320nm and 380 nm (Finkelstein,1994). The ultraviolet region of the electromagnetic spectrum is classified into three groups known as: Ultraviolet A (320nm-400nm), Ultraviolet B (290nm-320nm), and Ultraviolet C (200nm-290nm).

In the simplest way UVA rays excite psoralens and influence covalent bonding between pyrimidine bases of DNA (within skin cells) and the molecule. This creates a cross linkage creating an adductor; this molecule causes potential carcinogenic cells to form, which influence mutations and cell death (Bowers,1999; Diffey, 1991; Finkelstein, 1994; Rietschel, et al.1995). This is problematic as it can lead to weakening of cell walls, and cause cell death within animals. This is most detrimental when there is an interaction between furanocoumarins and UVA light incidence. (Zangerl et al., 1989; Rietschel et al., 1995). This can cause a hyperpigmentation of the skin, which are caused by the psoralens acting as photoprotection.

3.2 Negative Implications regarding Agriculture and Livestock:

Present analysis is scarce regarding negative impact of wild parsnip on livestock animals and agriculture. Fundamentally known to be present within forage fields and pastures, these invasive weeds can lessen overall yield and quality of the field, leading to potential pathogens causing problems within foraging animals.

Considering this if the presence of furanocoumarins in livestock diets have been shown to prevent mass gain and decreases the chances of fertility (Tassie and Kellie 2014). This is parallel to humans and the diet of omega 6 fatty acids. The major observational change implied with mammalian health and westernized diets are an increase in adipose tissues or fat tissues. Also, the furanocoumarins can cause PPD in the mouth of grazing animals. This can reduce life expectancy and be a negative impact economically to farmer (Ivie,1982).

4. Mitigation techniques

Kingston, as other Ontario municipalities are required to pass bylaws that address the presence of such “pests” when there is a risk of detrimental effects towards human health and safety of others (Tassie and Kellie 2014). This will allow for potential legislation changes, towards the by-laws because of the dangers regarding furanocoumarins. This is found under the legislation known as the Building Code Act. This act allows for Bill 64 to be overlooked when there is a risk towards human health and safety. One of these exceptions is regarding public health and safety when controlling: poisonous plants, insects, and organisms that can potentially damage structures.(https://news.ontario.ca/ene/en/2009/03/ontarios-cosmetic-pesticides-ban.html)

As well, The Invasive Species Centre of Ontario recommended a more futuristic approach towards mitigating invasive species. They have proposed conducts to mitigate wild parsnip. They suggest removal of future seedlings and follow-up monitoring of the negated area, also the removal of satellite populations to avoid further spread of wild parsnip. Secondly, they suggested focusing on the high-priority issues such as satellite populations and this is what is driving changes in ecosystems. As wild parsnip invades natural ecosystems, they cause change in landscapes such as nutrient availability in the soil. Lastly, they suggest re-planting native species, to restart native selection. This will reduce the invasive populations and allow for an increase in quality by removing the invasives. It will also allow for no-till farmers to continue their operations (Tassie and Kellie 2014).

4. 4. Herbicide control – Glyphosate

Glyphosate is an important means of mitigating harmful weeds in the world. This is a broad-spectrum foliar herbicide with high translocation (Franz et al. 1997; Duke, & Powles, 2008). Glyphosate inhibits the sixth enzyme in the shikimate biosynthetic pathway. This will prevent the essential nutrients and plant compounds such as aromatic amino acids from synthesis (Duke, and Powles, 2008). The problem when it comes to vegetative management with glyphosate is its lack of selectivity. Glyphosate is highly effective and therefore imposes high selection pressure for resistance when used repeatedly.  Glyphosate exponentially drains sources of carbon needed for growth. This inhibition induces these changes biologically and cause death. The problem with this comes from the fact it is a non-selective pesticide and can kill various plants. This ability to kill various plants has led towards resistances amongst populations. Glyphosate works by inhibiting EPSPS and this causes the production of chorismate, a compound used to produce primary aromatic compounds to hault.  This then stops the negative feedback loop of L-arogenate. This causes a Carbon drain on the plant and accumulation of shikimic tissues. This is what causes mortality in the plants.

5. Discussion and Methods:

During the summer of 2019, I collected Wild parsnip seeds from 18 randomized locations; across South Eastern Ontario. The dates are the basis for how I created these acronyms used in this experiment. The date July 26^th, 2019 was the first day of collection, and I have collected wild parsnip seeds from two locations. The first latitude and longitude collection took place in Tyendinaga Ontario. The latitude was 278 degrees West. The second latitude also took place in Tyendinaga and had 283 degrees West. Therefore, the coding is as follows SEO 1.1 is the first location I sampled from. The second location I collected seeds from on July 26^th is classified as SEO 1.2. The second date of collection occurred August 8^th, 2019 I collected from 5 random locations within Kingston and Napanee Ontario. This took place at Cataraqui woods drive, Fortune Drive and Parts of Southwood drive in Napanee. This would further be classified as SEO 2, and the following acronyms: 2.1, 2.2, 2.3, 2.4 ,2.5. These areas are of significance to me, as these locations are constantly covered, with wild parsnip.

The third date of collection occurred on August 9^th, 2019. This was random as I was sent into this location, again for work (I collected while I was on the clock at work dependent of time). I collected from 3 randomized locations that day. These were prominent in the Southwood Drive, Water Street, and lastly Camden road of Napanee Ontario. I classified these as SEO3. These were labelled as such; 3.1,3.2,3.3. This is an area of new development and I decided that wild parsnip here could have significance and decided to collect. The fourth collection was collected within Kingston and Bath Ontario. These were collected on August 12^th, 2019. I collected from 3 randomized locations during this collection. These took place at Fortune Drive, Taylor Kidd Drive and Country Club Drive in Kingston and Bath Ontario. The following is classified by the acronym SEO4, and ultimately follows the general pattern of 4.1, 4.2, 4.3 etc.

The fifth location occurred in Napanee on August 13^th, 2019. These took place at two locations known as, Switzerville Road, and Hartwood Drive. These were classified as SEO 5. These were labelled as 5.1, and 5.2. The wild parsnip had a warning sign around Hartwood drive this time. Also, it seemed to have been sprayed, by the town, with glyphosate on August 10^th, 2019. It was all vastly growing in a very; wet and clay soil environment. I collected, as I still noticed mature seeds visible within the wild parsnip populations. The sixth location occurred on August 14^th, 2019, and ultimately consisted of one collection considering that it was towards the end of the production season. Therefore, I did not collect from as many locations as would have liked. I essentially collected from two locations within Westbrook Ontario, near Leyton Drive, and lastly Water Street in Napanee. These were classified as SEO 6, this follows the general pattern as before, 6.1 is Leyton drive and 6.2 is water street. Lastly, the final collection occurred August 15^th, 2019. This occurred in Napanee Ontario on water street, near the utility plant. This was classified as SEO7 and labelled 7.1 as a result.This allowed less elevated wild parsnip seeds to be collected. This will allow variation in our collection which is what we want (randomized). Unfortunately, this was towards the end of seed dispersal and maturity for the perennial plant.

The project officially began September 3^rd, 2019. The first test trial was consisting of 2 black plant cell containers, with 6×3 dimensions. Each of the two black plant cells in each row were filled with various soil types. The types that were tested are: Turface, LA4, Promix, Sand and BGX.  The first plant cell was filled in the 3 rows by 3 plots completely. This consisted of the following soils, the first row was filled with Turface, the second row was filled with LA4, and third row was filled with Promix. The second plant cell was filled completely only 2 rows by 3 plots. This consisted of the following soils, the first row was filled with Sand and the second row was filled with BGX, the third row was left empty. The reasons for this test was to determine if soil type induced scarification on seeds. Wild parsnip is known to have grown in various soils and locations. Therefore, it was significant to test this, as the mesocarps could potentially be viable to scarification in any typical soil type. This could lead towards determining an optimal soil, for these seeds to grow. Each soil contained a seed from a various location of wild parsnip. These were collected and planted in these black plant cell containers. Two Wild parsnip seeds were planted in each cell. The soil type that had germination of cotyledons, at a better growth rates then other soils, was Promix. This treatment was given a secondary treatment of ecodormancy. This was carried out by placing both black plant cell containers inside the storage refrigerator; in the Crop Science Building. This test was to determine if ecodormancy was needed for these seeds to grow. This storage refrigerator was at 6 degrees Celsius and placed inside for 3 weeks. This was between September 6^th and September 24^th, 2019. Afterwards this trial was essentially stopped and not repeated as it took a longer duration then desired.

Another treatment was implemented while the two black plant cell containers were left in storage. This trial was done inside the Spray room (room #125 in Crop Science Building UOG) with potassium nitrate at 2% concentration. This trial was completed under a fume hood. This trial was completed to potentially test and see if this will allow a better alternative to acidification, and scarification of the mesocarp. This allows easier access for water to enter the seeds and initiate germination. The trial consisted of 2.00 grams of potassium nitrate added to 100.00mL of water. A weighing tray was initial put inside the balance and tared. The weighing tray was then removed from the balance and potassium nitrate was carried out, by using a spatula and adding it to the weighing tray. This was then weighed inside the balance. This was added to 100.00mL of water inside a 200.00mL flask. This was completed three times and essentially, we had three replicates. Therefore, there was three 200mL flasks with 100 mL of 2% potassium nitrate. A conversion of mass transfer was done and determined how many seeds will be added to each flask. It was determined that each flask contained 0.50 grams of seeds. This means each 200mL flask contained 100 seeds. The first two replicates consisted of two control cultivated parsnip varieties from; Ontario Seed Company and William-Dam Seeds. The third replicate consisted of wild parsnip seeds from location 4.1.  These were added to each 200mL flask and allowed saturation for 24hours. These seeds after 24 hours were removed from their flasks by draining the 2% potassium nitrate into a waste beaker. The waste beaker had a funnel with wired gauze over it. This will allow drainage and the seeds to be safely removed. Forceps were used and the seeds were added back into their original weighing trays. These weighing trays were used to transfer the seeds into plant cells. Two black plant cells (3cellsx3cells) were filled using the various soils methods listed above. Promix was the soil type used and we created seed banks. This was a hollow plant cell filled with Promix soil. Three seed banks were created; one for each parsnip seed type (OSC, WDS, SEO4.1). They contained 8.0 grams or, 400 potassium nitrate saturated seeds in each seed bank. This method and was continued moving forward. To determine the overall LT50 Value, it was estimated that we would test 112 seedlings between 56 pots. The 56 pots were divided into 28 pots for each cultivar. The first cultivar was from the Ontario Seed Company, the second cultivar is from the William-Dam Seeds. This would allow four replicates to be planted in each pot, with 7 dose concentration treatments to be tested. The concentrations we tested were as followed: 900g a.e./100ml, 450g a.e./100ml, 225g a.e./100ml, 112g a.e./100ml, 56g a.e./100ml, 28g a.e./10ml ,0g a.e./100ml. The highest rate on our log scale is 900 g a.e./100 ml. Therefore when 1.6ml of glyphosate is added to 100ml of deionized water, this allows the correct full dosage. When micro pipetting 1.6 ml of the glyphosate mixture into another 100ml of deionized water, this will allow the log rate of 450 g ae/ml to be created in the solution. The dilution of adding another 100mL of deionized water allows the dosage to be reduced by half. This process was repeated until we reached the concentration of 28 g a.e. /100ml.

These seven treatments were added one at a time into the spray tank with the nozzle. The angle of the nozzle was 90 degrees, and perpendicular to the plants. The angle would allow a greater coverage and direct contact with Glyphosate. This would allow us to see greater results of the pesticide working. The trolley was activated and allowed a dry run. The two treatments were done simultaneously for each various concentration. The plants were then allowed 5 minutes to dry inside the fume hood of the spraying apparatus. These treatments were then removed from the spraying apparatus and transported to Growth Room 7 (in the Crop Science Building); once they had dried. These were completed on November 9^th, 2019. The data was collected and analysed on November 24th, 2019.This will be used to create an LT50, which is the rate of applied glyphosate that kills half the population. This was analyzed and determined to be 56 g a.e./100ml of water.

The plants were then harvested from the first true leaves upward. These were collected from each pot, labelled by cultivar concentration of dosage and replicate. These were then placed in envelopes and put in the drier. These were dried at 60 degrees Celsius for 48 hours. The biomass was then collected from mass transfer and analysed. The overall significance from this was the William Dam-Seed cultivar, was more tolerant of glyphosate at the full dose of 900. The biomass was averaged to be 1.05025g, while the Ontario Seed Company was more susceptible. The Ontario Seed Company Cultivar had an average biomass at 900 of 0.3357175g.

6. Conclusion

The data determined that the LT50 is 56 g a.e /100 ml, which would then ultimately lead to us logically believing that; 112 g ae/100 ml is the rate that would kill the entire population. This is significant as this can be used as a rate to compare tolerance between a control and wild variety. We would need more trials to confirm this. Also, the biomass analysis allowed us to potentially see if certain cultivars are more tolerant than the other. In this case or findings point to that being correct. The biomass was more noticeable in the William Dam-Seed cultivar. This can tell us that this cultivar was more tolerant of glyphosate. This is because more biomass essentially means more carbon within the plant. This then allows more Carbon to be readily available, allowing energy to be spent on synthesis of foliage/biomass. Therefore, the dose of glyphosate at 900, is more susceptible for the Ontario seed company cultivar. This is because there is less biomass leading to think there is greater inhibition of EPSPS in the OSC seed cultivar. This is because as chorismate is inhibited there is no L-arogenate to complete the negative feedback loop causing an accumulation of shikimic tissue. This causes a Carbon drain in the plant and depletes the ability of the plant to synthesis amino acids and this depletes the plant as a result. Therefore, there will be less biomass as a result. Lastly, these findings seem to point in the direction that certain parsnip cultivars are more tolerant to glyphosate than anticipated. This is worthy of further testing to determine an answer. I would want to redo this experiment by increasing the chemical concentration.

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