Introduction

2.1.1 Variation in herbicide susceptibility

For the evolution of herbicide resistance to occur genetic variation in herbicide susceptibility must be present within a population. This variation may pre-exist as standing genetic variation, or it may be introduced via new mutations and/or gene flow (Jasieniuk et al. 1996). It can be assumed that even in naïve weed populations additive genetic and phenotypic variation for herbicide susceptibility will be present (Neve et al. 2014). Phenotypic variation is extremely important in determining how an organism responds to selection pressures, such as herbicide application (Hendry et al. 2011). Where this variation is heritable under selection pressure it can lead to a reduction in herbicide susceptibility in weed populations (Busi et al. 2013b). Therefore, understanding initial variation in pesticide susceptibility is important in investigating the early stages of resistance evolution, but has arguably been overlooked in the evolution of herbicide resistance (Neve et al. 2009).

Adaptation from standing genetic variation is predicted to occur over fewer generations than adaptation from new mutations, as the beneficial alleles are immediately available within the population (Barrett and Schluter, 2008). The initial frequency at which these alleles are present within the population will greatly affect the time taken for adaptation, with a high frequency leading to resistance evolution over fewer generations (Neve et al. 2003a). The frequency of alleles related to resistance will vary between populations both before and after herbicide exposure

(Ulber et al. 2013). In exposed populations where this variation in susceptibility is significant without the presence of resistance is it is possible that there is creeping resistance, where over a number of generations recombination of minor resistance alleles leads to a gradual shift towards resistance that goes unnoticed in the field (Gressel, 2009; Espeby et al. 2011, Chapter 1.3.5). If creeping resistance is occurring it can be predicted that the variability in susceptibility between weed populations will increase (Espeby et al. 2011). Collecting herbicide sensitivity data in weed populations can be extremely useful for detecting creeping resistance, as it can highlight its occurrence in exposed populations, or be used to detect changes in populations when exposed to new herbicides.

2.1.2 Sensitivity data

Establishing the sensitivity of weed populations to herbicides can be useful in detecting a shift towards resistance, as in the short term most adaptation is likely to arise from standing genetic variation and understanding this variation can provide an excellent indication of evolutionary potential (Hendry et al. 2011; Ulber et al. 2013). This initial variation has been reported in some species, for example, Espeby et al. (2011) found variable response to flupysulfuron in Alopecurus myosuroides populations, and to sulfsulfuron in Apera spica-venti populations, none of which had previous exposure to the herbicides. This baseline information can only be gained from populations that have not previously been exposed to the herbicide mode of action, of which there are now few.

However, the lack of untreated populations does not mean that there is no value in comparing sensitivity amongst populations, as this provides insight into the current

level of herbicide susceptibility and resistance in weed species (Ulber et al. 2013). For example, when testing populations that had previously been exposed to the herbicides, Espeby et al. (2011) also reported variable response and previously unreported resistance in populations of A. myosuroides to fenoxaprop-P-ethyl, which conferred cross resistance to flupysulfuron, and variable response in populations of Apera spica- venti to isoproturon, which did not confer cross resistance to sulfsulfuron.

2.1.2.1 Glyphosate sensitivity data

In contrast to baseline variability studies of unexposed populations and those where there is variation in exposed resistance populations variation in response to glyphosate has been reported in exposed populations of different species without the presence of resistance. Escorial et al. (2011) found variation and decreased glyphosate susceptibility in Bromus diandrus between different Spanish regions of 5.9% and 13.8%. Variation in the same populations to the herbicides chlortoluron, diclofop- methyl and chlorsulfuron was also reported. Boutin et al. (2010) reported variation in response to glyphosate in eight plant species collected from three to seven locations around the world, with GR25 values ranging from 60 to 98 g ha-1 for Bellis perennis L.

(English daisy), and 104 to 228 g ha-1 _{for Digitalis purpurea L. (common foxglove).}

There is also much variation in glyphosate sensitivity in species where resistant populations have already been reported. Loureiro et al. (2010) found 6.9% of Spanish Lolium rigidum populations tested for glyphosate resistance displayed intermediate or full resistance. Kniss et al (2007) reported a range of glyphosate susceptibility and resistance in USA Chenopodium album populations ranging between complete resistance to complete susceptibility at 840-glyphosate g ha-1_{, with a strong}

relationship between past glyphosate use and reduced sensitivity. When investigating two glyphosate resistant L. rigidum populations from perennial crops, Collavo and Sattin (2012) found ED50 values of 340 and 5319 g ha-1, with the latter population

having high levels of resistance conferred by multiple mechanisms.

There are only a few target site glyphosate resistance alleles know and it is likely that most mechanisms are non-target site (Yuan et al. 2007), which can be polygenic and under diverse control (Délye et al. 2013a). Therefore it is possible that variation in glyphosate susceptibility will have a major impact on non-target site resistance evolution. To date no glyphosate susceptibility studies have been reported in the UK. With variation in susceptibility to glyphosate and other herbicides reported in various countries and weed species and this variation in exposed populations being indicative of creeping resistance, variation in susceptibility is something that needs to be investigated and quantified to determine the state of glyphosate susceptibility/ tolerance in the UK.

2.1.3 Objectives

The main objective of this chapter is to investigate the variation in susceptibility of UK populations of A. myosuroides to the herbicide glyphosate. This is accomplished through dose-response analysis to determine variation in glyphosate susceptibility amongst a set of 55 A. myosuroides populations collected from around the UK (16 collected in 2010, and 39 collected in 2012).