Author ORCID Identifier

Date Available


Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Agriculture, Food and Environment


Plant and Soil Sciences

First Advisor

Dr. Michael Barrett

Second Advisor

Dr. Randy Dinkins


Incorporation of red clover (Trifolium pratense L.) into grass pastures offers several benefits. However, red clover cultivars available for Kentucky producers are highly susceptible to synthetic auxin herbicides, such as 2,4-D (2,4-dichlorophenoxy acetic acid), used for pasture broadleaf weed control. To overcome this problem, UK2014 red clover was bred for increased tolerance to 2,4-D. We compared the field performance of UK2014 to Kenland, a standard cultivar grown in the transition zone of the United States. UK2014 and Kenland were seeded in the springs of 2017 and 2018. Single applications of 0, 1.12, or 2.24 kg ae ha-1 2,4-D were made in June, August, or October. The red clover was harvested one week after the treatments and yield determined. Visual herbicide injury ratings were made prior to harvest and regrowth was visually assessed one week after harvest. Red clover stands were visually assessed the following spring. Kenland, across all application timings, was injured more by 2,4-D than UK2014 with mean injury ratings of 39% and 63% compared to 26% and 37% at 1.12 and 2.24 kg 2,4-D ae ha-1, respectively. At equivalent rates, Kenland regrowth was less than UK2014 at all application timings. UK2014 regrowth after 2,4-D treatment ranged from 65% to 91% while Kenland regrowth ranged from 12% to 72%. Applications of 2,4-D in October were the most damaging to stands of both UK2014 and Kenland the following spring, but Kenland stands were reduced much more than those of UK2014. Kenland and UK2014 had similar season total yields when not treated with 2,4-D (means of 7,550 and 7,880 dry matter kg ha-1, respectively, in 2017 and 5,280 dry matter kg ha-1 for both in 2018). Kenland season total yield in 2017 was reduced by both 2,4-D rates applied in June or August and at all timings in 2018. UK2014 season total yield in 2017 was only reduced when 2.24 kg 2,4-D ae ha-1 was applied in August. In 2018, 2.24 kg ae ha-1 2,4-D reduced UK2014 season total yield across application timings. UK2014 has greater 2,4-D tolerance than Kenland, but additional selection might be beneficial.

Further selection for 2,4-D tolerance of the UK2014 population produced the UK2020 population. We compared the 2,4-D tolerance of these two red clover populations to FL24D, a recently released 2,4-D tolerant red clover cultivar, Kenland, one of the parents of UK2014 and UK2020, and Southern BelleÔ, a known 2,4-D sensitive cultivar, in a greenhouse study. Based upon 2,4-D injury symptoms, plant biomass two weeks after 2,4-D application, and regrowth three weeks after initial harvest, UK2014, UK2020, and FL24D are 2 to 15 times more 2,4-D tolerant, depending on which specific parameter is assessed, than Kenland and Southern Belle. Based upon regrowth, UK2020 and FL24D are slightly more 2,4-D tolerant than UK2014 but equivalent to each other. An additional greenhouse study was conducted with Kenland and UK2014 pretreated or not with malathion (diethyl 2-dimethoxyphosphinothioyl sulfanylbutanedioate) two hours before 2,4-D application. Malathion pretreatment reduced UK2014 2,4-D tolerance to a level similar to that of Kenland. The metabolism of 14C 2,4-D in leaves of Kenland and UK2014, pretreated or not with malathion two hours before 14C 2,4-D application, was also examined. UK2014 metabolized 2,4-D more than Kenland without malathion pretreatment. Malathion pretreatment reduced 2,4-D metabolism in UK2014 to an amount equal to that of Kenland. Malathion pretreatment did not reduce the amount of 2,4-D metabolized in Kenland, although both UK2014 and Kenland produced a polar 2,4-D metabolite, or metabolites, that eluted at 4 minutes. Averaged across UK2014 and Kenland, malathion pretreatment increased the estimated 2,4-D half-life in the treated leaf from 50 to 118 hours. We demonstrated that UK2014 and UK2020 have increased 2,4-D tolerance similar to FL24D whose germplasm, along with Kenland, was the basis for selecting UK2014 and UK2020. The increased 2,4-D tolerance in UK2014 and UK2020, and presumably FL24D, is likely attributed to enhanced 2,4-D metabolism. Additionally, cytochrome P450 monooxygenases, one or more, are involved in the 2,4-D metabolism based on the results of studies including pretreatment with malathion, a known cytochrome P450 inhibitor.

We employed a transcriptome analysis approach to compare the gene expression response following 2,4-D treatment of UK2014 to that of Kenland in the field. There were two objectives. One, to determine if the increased 2,4-D tolerance in UK2014 is reflected in a change in transcriptional response and/or a quicker recovery of a transcriptional response following 2,4-D treatment. Second, to identify genes, whether constitutively expressed or induced by 2,4-D, that could be the basis for the increased 2,4-D tolerance. Leaf tissue from the two red clovers was collected at 4, 24, and 72 hours after 2,4-D (1.12 kg 2,4-amine a.e./ha) treatment from both untreated and treated plants. Global gene expression was determined with reads from Illumina Hiseq 2500 mapped against the red clover draft genome, Tpv2.1 (GenBank Accession GCA_900079335.1). Genes that displayed differential expression (DEGs) following 2,4-D treatment were selected for further analysis. The number of DEGs was higher for Kenland than for UK2014, showing that a lower transcriptional response corresponds with the higher 2,4-D tolerance in UK2014. Gene ontology enrichment analysis revealed that expression of photosynthesis related genes was affected less by 2,4-D in UK2014 than Kenland. Hence, we concluded that the increased 2,4-D tolerance of UK2014 correlates with a decreased transcription response to 2,4-D. However, we were not able to identify any specific genes that are the basis for the increased 2,4-D tolerance of UK2014. Nonetheless, several cytochrome P450 genes were highly expressed in both UK2014 and Kenland following 2,4-D treatment, suggesting these enzymes could be mediators of 2,4-D metabolism and tolerance in red clover.

Digital Object Identifier (DOI)

Funding Information

This research was supported by the Non-Assistance Cooperative Agreements 5864403005 and 5850428003 to Michael Barrett and by U.S Department of Agriculture (USDA) - Agricultural Research Service CRIS project 5042-21000-002-00D to Randy Dinkins, from 2016 to 2023.

This work was also supported by the Brazilian governmental scolarship "Ciência Sem Fronteiras", 222521/2014-7, awarded to Lucas Araújo by the National Council for Scientific and Technological Development (CNPq), from 2016 to 2019.

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Supplemental data for chapter 3

Supplemental excel File 2.xlsx (6181 kB)
Supplemental data for chapter 3

Available for download on Friday, August 02, 2024