Population sampling

To collect an annual sample of A. myosuroides seed from the seventeen fields, each

field was visited at the end of July/beginning of August in 2012, 2013 and 2014

when A. myosuroides seed was mature (Figure 3.1). A sample was collected from all

fields except those in which A. myosuroides was difficult to collect (e.g. certain, but

not all fields sown with Oilseed rape (Brassica napus)). To ensure samples were

random and representative of each A. myosuroides population, all tramlines in each

field were walked for the entire field. Seed was uniformly sampled by collecting one seed-head from plants at 10 metre intervals: a maximum of 500 seed-heads were collected per field. A bulk seed sample from each field was collected in paper bags, and bags were stored in a drying room (relative humidity = 15%) until use. At each

annual sampling, the presence of A. myosuroides across the field was scored on a

scale of 0-5: 0 = no black-grass found, 1 = a low density of occasional solitary plants, 2 = occasional small patches, 3 = large patches, 4 = widespread throughout the field, 5 = a dense and serious infestation.

3.3.2 Collection of weed management data

Information about the weed management applied to each of the seventeen fields was collected for an eight-year period between 2007 and 2014 (farmers for 4 fields failed to provide management information). The information collected included details of all crops sown and their sowing dates, the product names, dates and doses of all herbicides applied (glyphosate, pre-emergent and post-emergent herbicide) during

this period, and details of soil cultivation and any other cultural weed control practices during each year. This information was collected through a combination of farm visits, telephone and email conversations with farmers, and provided in the form of either photocopied written records or printed computer records.

Figure 3.1: A map of the 17 A. myosuroides populations sampled between 2011 and 2014. Points are positioned as close to the GPS co-ordinates of the field without obscuring any other. Populations are labeled as ALOMY_GBR_xx092, for example. For each sampling year - 2012, 2013, 2014 – the xx in each label is replaced by the year sampled, so the 2012 sample of ALOMY_GBR_xx092 becomes

ALOMY_GBR_12092. This method of labeling is used throughout.

3.3.3 Estimating phenotypic resistance frequency

After annual sampling, each seed population was phenotyped to estimate the proportion of individuals resistant to three doses of ALS and ACCase herbicide (0.5x 1x and 2x of the UK recommended field dose (1x) of each; a 0x control was also included for each MOA). The herbicides used were the ALS inhibitor mesosulfuron-

methyl + iodosulfuron-methyl-sodium (0.5x = 6 + 1.2 g.ai. ha-1, 1x = 12 + 2.4 g.ai.

ha-1_{, 2x = 24 + 4.8 g.ai. ha}-1_{) with the adjuvant Biopower (0.27 kg a.i ha}-1_{), and the}

ACCase inhibitor fenoxaprop-p-ethyl (0.5x = 41.5 g.ai ha-1, 1x = 83 g.ai ha-1, 2x =

herbicide*dose combination. Three standard susceptible populations were included – BR being susceptible to both ACCase and ALS modes of action, ALOMY_FRA07019 being ALS susceptible and ALOMY_GBH05001 being ACCase susceptible. Thirty two seeds were sown into each of 15 FP11 (L x W x H = 11x11x13cm) pots filled with a 2:1:1 mix of J. Arthur Bower’s topsoil (English loam blended with organic matter and nutrients, pH: 6.5 – 7.5), 0.5 Levington growing media: M2 (pH: 5.5 – 6, N: 200, P: 150, K: 200 mg/liter), and 0.25 J. Arthur Bower’s silver sand (lime-free washed silica sand). Individual pots were then placed into FP11 potholders, such that each potholder contained one pot for each population collected in a single survey year (Figure 3.2).

Figure 3.2: 15 pots, one for each population sampled in 2012, randomised within a potholder

Populations were randomly allocated positions in the potholder. Each potholder was randomly assigned a herbicide/dose combination and there were 15 potholders for each combination (total number of pot holders was 90, being 15 replicates*6 herbicide/dose combinations). Potholders were arranged in five blocks in a split plot design with main plots being blocks and sub plots being herbicide/dose combinations. Each block contained 3 herbicide/dose sub plots (potholders) and all

potholders were randomly arranged within a block. Pots were maintained in the

glasshouse (conditions: 22oC day/16oC night; 14 hour photoperiod) and regularly

watered until herbicide application; fertiliser was applied when required. Three weeks after sowing, plants were thinned to 16 plants per pot before being treated with the required dose of mesosulfuron-methyl + iodosulfuron-methyl-sodium or fenoxaprop-p-ethyl. Herbicide was applied using a Berthoud 2000 knapsack sprayer. The sprayer was set to apply herbicide at a pressure of 300 kPa while walking at a

fixed pace of 3 kph. A spray volume of 200L ha-1 _{was delivered through a Hypro}

standard flat fan tip (110 degrees F110-03 ultra-blue nozzle), positioned 40cm above the height of the modular tray. Plants were returned to the glasshouse compartment and watered regularly after herbicide application. Twenty-one days after herbicide application the number of plants surviving herbicide treatment was recorded. This protocol was followed for all three sampling years.

3.3.4 Estimating the frequency of target-site resistance and enhanced metabolism 3.3.4.1 Plant cloning

To determine the presence and frequency of target-site and/or enhanced metabolism mechanisms for ALS and ACCase resistance in annually resampled populations, approximately 25 seeds per sample were sown into a 8x8cm jiffy pot containing a soil mixture of loamy silt soil (LSI: 19% sand, 60% silt, 22% clay, 2.2% organic matter: pH 7.4), Once the seeds were sown, they were covered with approximately 0.5mm of Quartz sand RQ 16 (0.9-2.0 mm). Pots were arranged in polypropylene trays (L x W x H = 50x25x5cm), ten per tray and placed in a climate controlled glasshouse compartment. The glasshouse (temperature: 22.0°C/16°C; photoperiod: 14 hours; humidity: 50%) was equipped with supplementary lighting (turned on

when natural light intensity < 15 kilolux (bulbs: Philips Son-T Agro (400 W)) and an automatic shading system (activated when the light intensity exceeds 55 kilolux). All pots were watered via the trays as required. Once sixteen plants per sample had reached the three-tiller stage, plants were separated into individual tillers (clones). The cloning process involved removing the plant from the soil, separating the tillers at the shoots and roots using a scalpel, and cutting shoots to approximately 3cm in length. Cloned tillers were re-planted into fresh jiffy pots filled with the soil mixture described above and stood in fresh polypropylene trays. This process was repeated until each of the sixteen original plants had a minimum of five clones. Each of the five clones was used to identify the presence of ALS and ACCase target-site and enhanced metabolic resistance as described below.

3.3.4.2 Analysis of Target-site mutations and enhanced metabolism

For each of the sixteen plants per population sample, a 2cm section of leaf tissue was excised from the clone used as the untreated phenotyping control (see section 3.3.5.1) and the presence and frequency of target site mutations (two ALS and five ACCase) was assessed using the PCR and pyrosequencing method described in section 2.3.3. To assess the level of mesosulfuron (ALS) and fenoxaprop (ACCase) enhanced metabolism in each of the sixteen plants per annual population sample, two clones, one for mesosulfuron and one for fenoxaprop metabolism, were assessed using high-performance liquid chromatography using the method described in section 2.3.4.

3.3.4.3 Confirmation of resistance mechanisms by phenotyping

pyrosequencing endowed resistance, three of the five clones produced were reserved for phenotyping. One clone was treated with the UK recommended field dose of

mesosulfuron-methyl + iodosulfuron-methyl-sodium (12 + 2.4 g.ai ha-1_{) and the}

adjuvant Biopower (0.27 kg a.i ha-1), and one clone with the UK recommended field

dose of fenoxaprop-p-ethyl (83 g.aiha-1). The third clone was used as an untreated

control for comparison. There were 16 plants phenotyped for each population in each sample year. The 16 plants were arranged in four blocks within a glasshouse compartment, with 4 plants per population randomly assigned to each block. Plant*treatment combinations were randomly arranged in a fully randomised block

design. Herbicide was applied at a volume of 300 l ha-1 and a pressure of 200 kPa

using a track sprayer fitted with a Teejet XR8002 nozzle. The nozzle was positioned approximately 35 – 40 cm from the median plant height. After herbicide application,

5-6 ml (equivalent to approximately 100 kg N ha-1_{) of the liquid fertilizer (Wuxal}

Super: N8 – P8 – K6, citrus fertilizer) was applied. Twenty-one days after herbicide application, the mortality of every individual was determined it’s to establish resistance phenotype.

3.3.5 Data analysis and presentation