Michael Barrett

Incorporation of red clover (Trifolium pratense L.) into grass pastures can reduce the need for nitrogen fertilizer applications and increase the nutritional value of the forage. However, red clover cultivars available for Kentucky producers are highly susceptible to 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 selected for increased tolerance to 2,4-D. We employed a transcriptome analysis approach to compare the gene expression response following 2,4-D treatment of ‘UK2014’ to that of ‘Kenland’, a 2,4-D sensitive red clover and one of the parents of ‘UK2014’. The objectives were to first determine if the increased 2,4-D tolerance in ‘UK2014’ is reflected in a change of transcription response and/or a quicker recovery of a transcriptional response following 2,4-D treatment, and second, to identify genes, whether constitutively expressed or induced by 2,4-D, which could be the basis for the increased 2,4-D tolerance. Leaf tissue from the two red clovers grown in the field was collected at 4, 24, and 72 h after 2,4-D (1.12 kg 2,4-amine a.e. ha⁻¹) 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’, suggesting that a lower transcriptional response corresponds with the higher 2,4-D tolerance in the ‘UK2014’ line. Similarly, gene ontology enrichment analysis revealed that expression of photosynthesis-related genes was less affected by 2,4-D in the ‘UK2014’ line than ‘Kenland’. Although we were not able to identify any specific genes that are the basis for the increased 2,4-D tolerance of ‘UK2014’, we concluded that the increased 2,4-D tolerance of ‘UK2014’ correlates with a decreased transcription response to 2,4-D. Additionally, expression of several cytochrome P450 genes that had different isoforms between ‘UK2014’ and ‘Kenland’ increased significantly in both following 2,4-D treatment, one or more of these P450s could be mediators of 2,4-D metabolism and tolerance in red clover.

This study compared the field performance of red clover germplasm UK2014, selected for 2,4-D tolerance, to Kenland, a standard variety grown in the transition zone of the United States. UK2014 and Kenland were seeded in the spring of 2017 and 2018. Single applications of 0, 1.12, or 2.24 kg ae ha⁻¹ 2,4-D-amine were made in June, August, or October. One week after the treatments, yields were determined. Visible herbicide injury ratings were made prior to harvest and regrowth was visibly assessed 1 wk after harvest. Red clover stands were visibly assessed the following spring. Kenland, across all application timings, was injured by 2,4-D more than UK2014, with mean injury ratings of 39% and 63% compared with 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%, whereas 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 reduced only 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 resulted in reduced UK2014 season total yield across application timings. UK2014 has greater 2,4-D tolerance than Kenland, but additional selection might be beneficial.

Methiozolin is a new herbicide with an unknown mechanism of action (MOA) for control of annual bluegrass (Poa annua L.) in several warm- and cool-season turfgrasses. In the literature, methiozolin was proposed to be a pigment inhibitor via inhibition of tyrosine aminotransferases (TATs) or a cellulose biosynthesis inhibitor (CBI). Here, exploratory research was conducted to characterize the herbicide symptomology and MOA of methiozolin. Arabidopsis (Arabidopsis thaliana L.) and P. annua exhibited a similar level of susceptibility to methiozolin, and arrest of meristematic growth was the most characteristic symptomology. For example, methiozolin inhibited A. thaliana root growth (GR₅₀ 8 nM) and shoot emergence (GR₈₀ ˜50 nM), and apical meristem growth was completely arrested at rates greater than 500 nM. We concluded that methiozolin was neither a TAT nor a CBI inhibitor. Methiozolin had a minor effect on chlorophyll and alpha-tocopherol content in treated seedlings (Lemna aequinoctialis Welw.; syn. Lemna paucicostata Hegelm.) showed that methiozolin also reduced fatty-acid content in Lemna with a profile similar, but not identical, to cinmethylin. However, there was no difference in fatty-acid content between treated (1 µM) and untreated A. thaliana seedlings. Methiozolin also bound to both A, thaliana and L. aequinoctialis FATs in vitro. Modeling suggested that methiozolin and cinmethylin have comparable and overlapping FAT binding sites. While there was a discrepancy in the effect of methiozolin on fatty-acid content between L. aequinoctialis and A. thaliana, the overall evidence indicates that methiozolin is a FAT inhibitor and acts in a similar manner as cinmethylin.

Although the problem of herbicide resistance is not new, the widespread evolution of glyphosate resistance in weed species such as Palmer amaranth (Amaranthus palmeriS. Wats.), common waterhemp (Amaranthus rudisSauer), and kochia [Kochia scoparia(L.) Schrad.] raised awareness throughout the agricultural community of herbicide resistance as a problem. Glyphosate-resistant weeds resulted in the loss of a simple, single herbicide option to control a wide spectrum of weeds that gave efficacious and economical weed management in corn (Zea maysL.), soybean [Glycine max(L.) Merr.], and cotton (Gossypium hirsutumL.) crops engineered for tolerance to this herbicide and planted over widespread areas of the South and Midwest of the United States. Beyond these crops, glyphosate is used for vegetation management in other cropping systems and in noncrop areas across the United States, and resistance to this herbicide threatens its continued utility in all of these situations. This, combined with the development of multiple herbicide-resistant weeds and the lack of commercialization of herbicides with new mechanisms of action over the past years (Duke 2012), caused the weed science community to realize that stewardship of existing herbicide resources, extending their useful life as long as possible, is imperative. Further, while additional herbicide tolerance traits are being incorporated into crops, weed management in these crops will still be based upon using existing, old, herbicide chemistries.

Greenhouse and laboratory experiments were conducted to determine the basis of imazethapyr tolerance in cowpea. Cowpea required more than 700 g ha⁻¹imazethapyr to reduce shoot growth 50% while corn required 70 g ha⁻¹to reduce growth 50% in greenhouse experiments. Extractable acetohydroxy acid synthase activity from cowpea 24 h after foliar imazethapyr treatment was only 20% of that of untreated cowpea but increased to control levels 72 h after treatment (HAT). The unifoliate leaves of cowpea absorbed 25% of the¹⁴C-imazethapyr applied 72 HAT. Movement of¹⁴C-imazethapyr out of the treated zone increased gradually to approximately 47% of the absorbed¹⁴C 168 HAT. Parent imazethapyr decreased from 41 to 27% of the recovered¹⁴C between 4 and 24 HAT, and continued to decrease to 14% by 168 HAT. Polar metabolites increased from 27 to 55% of the recovered¹⁴C from 24 to 168 HAT. The polar metabolites of imazethapyr included hydroxyimazethapyr and conjugates of hydroxyimazethapyr. Tolerance of cowpea to imazethapyr is based on the plants ability to metabolize the herbicide to nontoxic metabolites.