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Characterisation of a Capsella bursa- pastorisbiotype resistant to ALS inhibitors

Master thesisAarhus University

Agrobiology - Plant Nutrition and Health

Antje Reiss

November 30, 2014

Supervisors:Professor Per Kudsk

Senior Scientist Solvejg K. Mathiassen

Characterisation of a Capsella bursa- pastorisbiotype resistant to ALS inhibitors

Karakterisering af en Capsella bursa- pastoris biotype resistent

overfor ALS inhibitorer

45 ECTS pointsMaster thesis

Aarhus University

Agrobiology - Plant Nutrition and Health

Antje Reiss

November 30, 2014

Supervisors:Professor Per Kudsk

Senior Scientist Solvejg K. Mathiassen

Summary

The main factor reducing potential yield worldwide are weeds. Therefore, weed resis-tance development to herbicides is one of the major threats conventional agriculturefaces today. Especially resistance to acetolactate synthase (ALS) inhibiting herbicidesincreased dramatically over the last two decades. Recently, a new point mutation inthe ALS gene at Trp574, leading to a substitution of tryptophane with serine, wasfound in a population of Capsella bursa- pastoris (CAPBP) in Denmark. In thisthesis the progeny of this resistant population, biotype ID1120 and ID1123, were char-acterised in their phenology and morphology and compared with susceptible biotypeswith different geographic origin. Earlier point mutation analysis gave evidence forthe presence of Trp574-Ser in ID1120 and absence in ID1123. Therefore, the presenceof point mutations in ID1123 was analysed in a genetic analysis. A competitionexperiment was conducted to identify possible cross resistance patterns or multipleresistances to other herbicide modes of action. Further, possible fitness costs due toresistance were investigated with competition and germination experiments.

It was evident that the present biotypes resembled the diversity present in thespecies CAPBP. In accordance to this, there were no significant differences betweenresistant and susceptible biotypes concerning phenological and morphological traits.It was not possible to identify point mutations in ID1123 with the genetic analysisconducted. In the dose- response experiment ID1120 was resistant to florasulam,flupyrsulfuron and iodosulfuron but not to imazethapyr, thereby conferring crossresistance to triazolopyrimidines and sulfonylureas. This resistance pattern differedfrom other cases of Trp574 resistance which conferred broad cross resistance, includingresistance to imidazolinones. ID1123 was only resistant to sulfonylureas and there wasno evidence for multiple resistance in either biotype. It was concluded that ID1123had a different resistance mechanism than ID1120. The nature of this resistancemechanism is unknown and it can not be excluded that it is also present in ID1120.The analysis of fitness costs due to resistance resulted in the identification of alteredtotal germination of both resistant biotypes. At high temperatures there was evidencefor a fitness cost and at low temperatures a fitness advantage of the resistant biotypes.

This characterisation of resistant CAPBP biotypes with the recently found pointmutation Trp574-Ser shows, that there is still potential for new mutations conferringresistance to be found and that they might provide unpredictable cross resistancepatterns.

e

Preface

This thesis completes my master studies in Agrobiology, Plant Nutrition and Healthat Aarhus University. It is a 45 ECTS points thesis, conducted from April to the endof November 2014 at the Research Centre Flakkebjerg, department of Agroecology.The practical work contained experiments in the greenhouse, semi field area andlaboratory. The experiments were planned in collaboration with my supervisorsProfessor Per Kudsk and Senior Scientist Solvejg Kopp Mathiassen and executed andanalysed under my own responsibility.

At this point I want to thank Professor Per Kudsk for giving me the opportu-nity to work on this project. Furthermore I want to thank Senior Scientist SolvejgKopp Mathiassen for her help during the whole period and especially during thewriting process. I also want to thank Betina Bendtsen and Christian Appel SchjeldahlNielsen for taking care of my plants and helping me with the practical work. Inaddition I want to thank Marielle Babineau for her tips in the laboratory and forcritically reading this thesis. Last but not least I want to thank my office mates, myfellow students and especially the "weed group" for the inspiring working atmosphere,talks, help and good advice.

Flakkebjerg, 30.11.2014

Antje Reiss

g

Contents

1 Introduction 1

2 Theory 52.1 The species Capsella bursa-pastoris . . . . . . . . . . . . . . . . . . . 52.2 Acetolactate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Regulation mechanism . . . . . . . . . . . . . . . . . . . . . . 62.3 Resistance mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.3.1 Target site resistance . . . . . . . . . . . . . . . . . . . . . . . . 8Herbicide binding . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.3.2 Metabolic resistance . . . . . . . . . . . . . . . . . . . . . . . . 92.3.3 Fitness cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Fitness cost due to target site resistance . . . . . . . . . . . . . 9Fitness cost due to metabolic resistance . . . . . . . . . . . . . 10

3 Materials and methods 113.1 Overview of the seed material used . . . . . . . . . . . . . . . . . . . 113.2 Herbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.3 Characterisation experiment . . . . . . . . . . . . . . . . . . . . . . . 14

3.3.1 Pot experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 143.3.2 Germination experiments . . . . . . . . . . . . . . . . . . . . . 15

3.4 Dose-response experiment . . . . . . . . . . . . . . . . . . . . . . . . 153.4.1 Post emergence herbicides . . . . . . . . . . . . . . . . . . . . . 153.4.2 Pre emergence herbicide . . . . . . . . . . . . . . . . . . . . . . 16

3.5 Competition experiment . . . . . . . . . . . . . . . . . . . . . . . . . 183.6 Genetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.6.1 DNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 183.6.2 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . 193.6.3 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.7 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4 Results 234.1 Characterisation experiment . . . . . . . . . . . . . . . . . . . . . . . 23

4.1.1 Pot experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Correlogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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ii Contents

Phenological traits . . . . . . . . . . . . . . . . . . . . . . . . . 23Morphological traits . . . . . . . . . . . . . . . . . . . . . . . . 26Leaf type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4.1.2 Germination experiments . . . . . . . . . . . . . . . . . . . . . 29Germination conditions . . . . . . . . . . . . . . . . . . . . . . 29Comparison of germination of the CAPBP biotypes used in the

characterisation experiment . . . . . . . . . . . . . . . 324.2 Dose- response analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 334.3 Competition experiment . . . . . . . . . . . . . . . . . . . . . . . . . 384.4 Genetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

5 Discussion 43Characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . 43Leaf type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Dose- response experiment . . . . . . . . . . . . . . . . . . . . 46Competition experiment . . . . . . . . . . . . . . . . . . . . . . 48Plant fitness . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Genetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 50

6 Conclusion 51

7 Perspectives 53

Bibliography 55

Appendix 65

A First chapter of appendix 65A.1 Characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

A.1.1 Characterisation experiment one . . . . . . . . . . . . . . . . . 65Morphological traits . . . . . . . . . . . . . . . . . . . . . . . . 65

A.1.2 Characterisation experiment two and three . . . . . . . . . . . 66A.1.3 Germination experiment one . . . . . . . . . . . . . . . . . . . 72

A.2 Dose-response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Contents iii

CHAPTER 1Introduction

Weeds are the main reason for potential yield loss in conventional agricultural systemsworldwide. While weeds account for a potential yield loss of 34%, animal pestsand pathogens cause only 18 and 16 %, respectively (Oerke, 2006). In contrast tothis, the efficacy of weed control is with 75% almost twice as effective as control ofpathogens and animal pests with 32 and 39%, respectively (Oerke, 2006). This largeprofit is mainly thanks to the use of herbicides, supplementing other weeding practices.

Acetolactate synthase (ALS) inhibiting herbicides are a widely used herbicide groupdue to their broad spectrum weed control, low mammalian toxicity and wide cropselectivity (Yu et al., 2014). However, herbicide resistance developed very rapidlyafter its introduction and the first case was confirmed in Lolium rigidum in 1986(Heap et al., 1986; Heap et al., 1990; Yu et al., 2014). When the first case ofALS resistance was found in Europe in 1991 (Kudsk, Mathiassen, et al., 1995),the number of new cases already increased exponentially worldwide (figure 1.1).To date, a total of 145 species is resistant to ALS inhibiting herbicides, hereof 88dicotyledonous species (Heap, 2014). This development is not as dramatic for otherherbicide modes of action, but also here the number of resistant cases is increasing(figure 1.1) (Heap, 2014).

Once resistance developed, the affected herbicide cannot be used for effective weedcontrol in the area where the resistant weed occurs. Dependent on the resistancemechanism also cross resistance and multiple resistance may be present, excludingone or even multiple modes of action. However, the resistance alleles may havepleiotropic effects on plant fitness (Vila-Aiub et al., 2009[b]). Hereby the magnitudeof fitness cost is negatively correlated with the possibility that resistance alleles arefixed in the population. This means that if high fitness costs are present, the numberof resistant individuals in the population may decline over time under conditionswhere the herbicide is absent (Vila-Aiub et al., 2009[b]). But if no fitness costs areassociated with the resistance mechanism, resistance will stay. The majority of casesof target site resistance to ALS inhibiting herbicides only show subtle fitness costs and

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2 1 Introduction

Figure 1.1: Resistance development of herbicides with different modes of action (Heap,2014)

fitness costs due metabolic resistance were, to current knowledge, not investigated ata dicotyledonous species (Vila-Aiub et al., 2009[b]; Yu et al., 2014).

The ineffective modes of action could theoretically be replaced by new herbicides.Unfortunately, the amount of new compounds that are released to the market isdeclining and nearly stagnated (Kudsk and Streibig, 2003). The discovery ofpyrasulfotole in 2008 was the first significant new mode of action for use in cerealssince more than twenty years (Reddy et al., 2013). This is of concern to conventionalcropping systems, as especially noninversion tillage systems rely on herbicides forweed control to maintain current cost-performance ratio (Chauhan et al., 2012;Melander et al., 2013). Consequently, much effort is done to prevent or at leastdelay resistance development. A reasonable strategy is the implementation of inte-grated weed management by combining the usage of mixtures of herbicides of differentmodes of action (Beckie et al., 2009; Streibig et al., 1998) with non-chemical toolssuch as mechanical weeding (Kudsk and Streibig, 2003). Exchange of modes ofactions is getting increasingly difficult in Europe, because Directive 91/414/EEC,the Pesticide Authorisation Regulations EC/1107/2009 and the Water FrameworkDirective (2000/60/EC) led to a decrease of available herbicides on the market. InDenmark, pesticide control actions were already implemented in 1986 (summarised byHaas et al., 1994) and the consequences for the Danish weed flora were analysed by

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Andreasen and Stryhn, 2012. The species Capsella bursa-pastoris (CAPBP) is oneof the species that remarkably increased in frequency (from 1989-2001) (Andreasenand Stryhn, 2012). It is a key species for ecological diversity, physiologically andgenetically diverse and a frequently occurring weed species worldwide (Aksoy et al.,1998; Andreasen and Stryhn, 2012; Hanzlik et al., 2012; Hawes et al., 2005).

Also in CAPBP, resistance to ALS inhibiting herbicides has been identified. Sofar, five cases of resistance of CAPBP to ALS inhibitor herbicides are recorded world-wide. The first resistant population was found in Israel in the year 2000, followedby two cases in Canada (2008, 2011), one in China (2009) and the most recent inDenmark (2012) (Heap, 2014). The exact resistance mechanism is only known for theresistant populations found in China and Denmark. In both cases there is evidencefor target site resistance. In the population from China, several point mutations atamino acid position Pro197 were identified (Cui et al., 2012; Jin et al., 2011; Wanget al., 2011). For the Danish population, it was the first time that a mutation atamino acid position 574, leading to a substitution of tryptophane with serine, wasidentified in a weed species. Both resistant biotypes characterised in this thesis areprogeny of this population found in Denmark. However, in only one of them (ID1120),the point mutation at Trp574-Ser was identified. The resistance mechanism of theother biotype (ID1123) remains unclear.

The aim of this thesis is to fully characterise two resistant biotypes of CAPBPand put this into perspective by comparing them to a selection of susceptible bio-types. For this purspose, several experiments were designed: The characterisationexperiment aims to fully describe the biotypes in their morphological and phenolog-ical characteristics. In the dose-response experiment cross resistance and multipleresistance patterns of the resistant biotypes are investigated and the competitionexperiment is designed to identify possible fitness costs attributed to resistance. Inaddition to this a polymerase chain reaction is conducted to provide a more detailedknowledge about the mechanism of the resistance.

On the basis of the large variation in the genotype, as well as the phenotype of thespecies CAPBP, the following hypotheses are formulated:

1. Morphological and phenological traits of resistant biotypes do not differ signifi-cantly from susceptible biotypes.

2. The present target site resistance leads to cross resistance patterns, but not tomultiple resistance.

3. There are no observable fitness differences between resistant and susceptiblebiotypes.

CHAPTER 2Theory

2.1 The species Capsella bursa-pastorisCAPBP belongs to the family brassicaceae, the tribe Camelineae and the genusCapsella. It is a self compatible, tetraploid species with a possibly allopolyploidorigin and worldwide distribution (Hurka, Friesen, et al., 2012). In Denmark, it iswith a frequency of nearly 32% in spring barley, 27% in winter barley and 22.5% inwinter wheat one of the most dominant weed species (Andreasen and Streibig,2011; Andreasen and Stryhn, 2008; Andreasen and Stryhn, 2012). The Danishdecision support system for farmers recommends six different modes of action forcontrol of CAPBP at 3-4 leaf stage in spring barley (according to HRAC: B,O,F,C+M,K) (plantevaern-online, 2014). In winter wheat the choice is more restricted.Here it is only two recommended modes of action to control CAPBP at 3-4 leaf stage:B and F in the autumn and B and O in spring (plantevaern-online, 2014). Thisshows that dependent on the crop it can be difficult for the farmer to find alternativesto ALS inhibiting herbicides for weed control.

Fig. 2.1: Four leaf types of thespecies CAPBP, defined by Shull,1909, whereas a) simplex, b) rhom-boidea, c) tenuis, d) heteris. Thefigure is from Iannetta et al.,2007.

Even though CAPBP is mostly a spring an-nual (Baskin et al., 2004), it can be foundflowering from late spring to late autumn(Linde et al., 2001). In some cases CAPBPmay develop frost tolerance, making a shiftfrom annual to biannual possible (Iannetta,2011).

The species CAPBP is generally very diverse andmuch research was done on characterising distinctbiotypes (Aksoy et al., 1998; Iannetta et al.,2007; Neuffer, 2011; Neuffer and Hurka,1988; Shull, 1909). Shull [1909] was the firstto distinguish between four different leaf types,simplex, rhomboidea, tenuis and heteris (see fig-ure 2.1). In the leaf form simplex, sinuses never

5

6 2 Theory

reach the midrib and it comprises of mostly simple rounded, not attenuated lobes.The rhomboidean leaf type is divided into the midrib and the terminal lobe is set offfrom the nearest lateral lobes. Tenuis is characterised by relatively shallow sinusesand all lateral lobes are slender, elongated and acute. Finally, heteris comprisesof leaves that are divided into the midrib, the terminal lobe is separated from thenearest lateral lobes by deep, clean cut incisions and the lateral lobes have a roundedportion close to the midrib and an elongated, attenuate portion at the tip.

Dividing the species CAPBP into these four leaf types is still accepted (Neuf-fer, 2011; Neuffer and Hurka, 1988). However, there are also attempts to findcontinual ways of defining leaf types, as the species is so diverse. For example,Iannetta et al., 2007 showed that, with the usage of genetic markers, a furtherdivision in seven leaf types is possible, confirming a broad diversity in the speciesCAPBP. However, in this thesis leaf types are determined visually after Shull, 1909.

2.2 Acetolactate synthaseThe acetolactate synthase is the common enzyme to the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine (Duggleby and Pang, 2000; Um-barger, 1978). It catalyses the first step of the biosynthetic pathway, the reactionof pyruvate. An overview of ALS catalysed steps is shown in figure 2.2 C. In thefirst step, common to all three branched- chain amino acids, ALS bound thiaminediphosphate (ThDP) reacts with the substrate pyruvate, releasing CO2 and formingenzyme bound hydroxyethyl-ThDP (Duggleby, McCourt, et al., 2008) (see figure2.2 A and B). Subsequently, the ALS catalyses two different reactions. One is the re-action of hydroxyethyl-ThDP with 2-ketobutyrate, leading to the further biosyntheticpathway of isoleucine (figure 2.2 A). Or the reaction of enzyme bound hydroxyethyl-ThDP with another pyruvate (figure 2.2 B), commencing the biotynthesis of leucineand valine (Duggleby, McCourt, et al., 2008). The further synthesis of valine andisoleucine is parallel and catalysed by the same three enzymes: ketol-acid reductoiso-merase (KARI) , dihydroxyacid dehydratase (DH) and transaminase (TA) (figure2.2 C).

For the synthesis of leucine, 2-keto-isovalerate from valine biosynthesis goes into aseparate pathway, catalysed by another three additional enzymes: 2-isopropylmalatesynthase (IPMS), isopropylmalate isomerase (IPMI) and 3-isopropylmalate dehydro-genase (IPMD) (figure 2.2 C) (Duggleby and Pang, 2000).

Regulation mechanismALS is feedback inhibited by the branched amino acids valine, leucine and isoleucing,with valine being the most potent inhibitor (Duggleby and Pang, 2000; McCourtet al., 2006). There is contrasting results about ALS being both competitively andnon-competitively inhibited in respect to pyruvate and the inhibition can range from89% to 13% (McCourt et al., 2006).

2.2 Acetolactate synthase 7

A

C

CH3

COO−

O

pyruvate

CO2

C

CH3

H

O

ThDPenzyme boundhydroxyethyl

+ C

CH2

CH3

COO−

O

2-ketobutyrate

C

CH2

CH3

COO−

C

O

H3C OH

2-aceto-2-hydroxybutyrate

B

C

CH3

COO−

O

pyruvate

CO2

C

CH3

H

O

ThDPenzyme boundhydroxyethyl

+ C

CH3

COO−

O

pyruvate

C

CH3

COO−

C

O

H3C OH

2-acetolactate

C

2-ketobutyrate + pyruvate

ALS

2-aceto-2-hydroxy-butyrate

KARI

2,3dihydroxy-3methyl-valerate

DH

2-keto-3-methylvalerate

TA

isoleucine

pyruvate + pyruvate

ALS

2-acetolactate

KARI

2,3dihydroxy-isovalerate

DH

2-keto-isovalerate

TA

valine

2-keto-isovalerate + acetyl-CoA

IPMS

2-isopropylmalate

IPMI

3-isopropylmalate

IPMD

2-keto-isocaproate

TA

leucine

Figure 2.2: Pathway of ALS biosynthesis.Figure A and B shows the two reactions, catalysed by ALS. Figure C shows the biosynthesispathway of the three branched amino acids isoleucine, valine and leucine. For further explanation,see the text. The figure was made after (Duggleby, McCourt, et al., 2008; Duggleby andPang, 2000).

8 2 Theory

2.3 Resistance mechanismsResistance mechanisms can be divided into target site resistance (TSR), defined as"changes at the target site that limit herbicide impact" (Delye et al., 2011; Powleset al., 2010) and non target site resistance (NTSR), defined as "mechanisms thatminimize the amount of active herbicide reaching the target site" (Powles et al.,2010). NTSR mechanisms are for example decreased herbicide penetration intothe plant, decreased herbicide translocation in the plant and increased herbicidemetabolism (Powles et al., 2010). When speaking about NTSR in the following, itis always referred to the mechanism of increased metabolism.

2.3.1 Target site resistanceIn the case of ALS target site resistance, so far multiple point mutations that lead toa change in eight amino acids have been reported. These amino acids are Ala122,Pro197, Ala205, Asp376, Arg377, Trp574, Ser653 and Gly654 (figure 2.3) (Tranel,Wright, and Heap, 2014). Hereby, Pro197 and Trp574-Leu are the most commonmutations observed (Powles et al., 2010). Different point mutations at Pro197in CAPBP were identified in a population from China in 2009 (Cui et al., 2012).Recently a mutation at Trp-574, leading to a substitution of tryptophane with serine,was found in a CAPBP population in Denmark. This is the first time that this partic-ular point mutation was found in a weed species (Tranel, Wright, and Heap, 2014).

Herbicide bindingBoth, SU and IMI bind within the tunnel leading to the active site of ALS andthereby block substrate access. In the tunnel, they have overlapping binding sites,sharing ten amino acids, including Trp574 (McCourt et al., 2006). These bind-ing characteristics lead to specific cross resistance patterns for the affected aminoacids, reflecting herbicide use patterns. Exclusive usage of SU herbicides selects for

Figure 2.3: Target site mutations conferring resistance to ALS inhibiting herbicides.Taken from Heap, 2014. Pro197 32%, Trp574 28%, Ala122 8%, Asp376 7%, Ser653 7%,Ala205 4%, Arg377 1%, Gly654 1%

2.3 Resistance mechanisms 9

Pro197 and mixed usage of SU and IMI herbicides selects for Trp574 point mutations(Powles et al., 2010). That the binding position is located not directly at the activecenter of the enzyme results in both herbicide groups being uncompetitive or mixedinhibitors with respect to the substrate pyruvate. Some sulfonylureas have also beenreported to be nearly competitive inhibitors (McCourt et al., 2006).

2.3.2 Metabolic resistanceMetabolic resistance is often characterised by the presence of multiple resistances(Yu et al., 2014). There are a number of reactions and associated enzymes identifiedas being part of the chemical modification and degradation of herbicides. The mostimportant include hydroxylation or dealkylation by cytochrome P450 monooxyge-nases, glutathion conjugation by the glutathion-S-transferase and conjugation withsugars by glycosyl-transferases (Delye et al., 2011; Yu et al., 2014). These reactionsmake it possible for the plant to detoxify the herbicide and transport it into thevacuole and/or the cell walls where further degradation may occur (Delye et al.,2011; Yuan et al., 2007).

2.3.3 Fitness costGenerally, there are at least three explanations for fitness costs due to herbicideresistance (Vila-Aiub et al., 2009[b]): One is that the resistance mechanism inter-feres with normal plant function or metabolism, for example by a point mutationcompromising enzyme function and kinetics. A second explanation is the possibilityof a trade- off between plant reproduction, growth and defence functions (includingherbicide resistance). The third possibility is altered ecological interaction, for in-stance reduced attractiveness to pollinators or increased susceptibility to diseases.All of these fitness costs may be environment-specific and may only be manifested atcertain lifehistory stages (Neve et al., 2009).

Fitness cost due to target site resistanceMany studies analysing possible fitness costs due to target site mutations confer-ring herbicide resistance to ALS inhibitors have been conducted (Vila-Aiub et al.,2009[b]). However, many of them are not easily interpretable due to a lack of controlof the genetic background and missing knowledge of the biochemical basis of herbicideresistance. Other factors that should be evaluated when analysing plant fitness are lifehistory traits, such as plant fitness cost during germination and plant establishment,resource competition and fitness costs in an environmental gradient (Vila-Aiubet al., 2009[b]).

Vila-Aiub et al., 2009(b) reviewed a large amount of studies investigating alternatedplant fitness due to ALS target site resistance and concluded that "the impact of eachspecific mutation/ amino acid substitution needs to be evaluated on a case-by-casebasis". Therefore, the focus here is on Pro197 and Trp574 mutations, as they are theonly two mutations so far confirmed in CAPBP (Tranel, Wright, and Heap, 2014).

10 2 Theory

Out of the various possible Pro197 mutations (see Tranel, Wright, and Heap,2014), fitness costs have been reported only for Pro197-His. Additionally, Trp574-Pheresults in fitness cost in form of reduced ALS activity (in tobacco) (Vila-Aiub et al.,2009[b]). The mutations Pro197-Ser and Thr574-Leu result in higher ALS activityand for Pro197-His and Pro197-Thr a decreased feedback inhibition by branchedchain amino acids was reported. This may lead to increased concentrations of valine,leucine and isoleucine in plant tissue, which was found to be correlated with higherseed germination rates at low temperatures (Dyer et al., 1993). If this results in afitness cost or advantage depends on the environmental conditions present. Unlessthat there are some individual plants where significant fitness costs could be identified,taken all investigated cases into consideration TSR has rather subtle effects on plantfitness (Yu et al., 2014).

Fitness cost due to metabolic resistanceSo far, there is only few studies on fitness costs due to metabolic resistance (Vila-Aiub et al., 2009[b]). All studies on fitness cost of P450 based enhanced herbicidemetabolism were conducted on Lolium rigidum and show decreased fitness of resistant,compared to susceptible biotypes. These costs are 20% reduction of vegetative growth,compared to the susceptible standard (Vila-Aiub et al., 2005). In a competitionexperiment with wheat, even 30% reduction in vegetative and 23% reduction inreproductive stage were observed (Vila-Aiub et al., 2009[a]).

These are the only two studies on fitness costs caused by metabolic resistanceto ALS inhibitor herbicides, reported to date.

CHAPTER 3Materials and methods

3.1 Overview of the seed material usedIn order to be able to set the characteristics of the resistant biotype into perspective,four susceptible biotypes with different origins were used. An overview of their originand resistance characteristics is presented in table 3.1.

Table 3.1: Overview over the seeds used in the experiments. The origin of seeds, aswell as its resistance status to ALS inhibitor herbicides is stated.

ID Resistance Seed originID140 S FlakkebjergID1117 S Herbiseed, Catalogue nr. 31186ID1118 S B & T Seeds, Catalogue nr. 31186ID1089 S Ringsted, KU Life, Prof. StreibigID1120 R (Trp574) Flakkebjerg, from ID970ID1123 R Flakkebjerg, from ID970, unknown mutation

The seed sample of ID970 originated from a field in northern Jutland, Denmark.This field was treated with SU herbicides in four out of five preceding years beforesuspicion on resistant CAPBP plants was raised. The seeds of ID970 were collectedfrom multiple individuals, which survived herbicide treatment with SU herbicides.Previously to this thesis, ID970 was analysed for resistance to the active ingredientsflorasulam and tribenuron in pot experiments and individuals were found to beresistant with an resistance index (RI) 4<RI<10 and of 75, respectively. Altogethersix plants survived tribenuron treatment with 1 g ai/ha which was proven to belethal to a susceptible standard population. The two seed lines ID1120 and ID1123are progeny of seeds from two of these individual plants of ID970. These six plantswere sent for analysis of point mutations at position Pro197 and Trp574. For noneof them, a mutation at Pro197 was identified. Though, one out of the six had amutation at Trp574, leading to substitution of the amino acid tryptophane with

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12 3 Materials and methods

serine. Progeny of this plant is ID1120 while ID1123 is progeny of another plantwhich did not have a point mutation at either Pro197, or Trp574. Due to their differ-ential characteristics, they are referred to as individual biotypes throughout this thesis.

3.2 HerbicidesThe herbicides used for dose-response analysis were chosen with the purpose toinvestigate cross resistance patterns of the resistant biotypes to several classes of ALSinhibitors. Additionally, some herbicides with other modes of action were chosen totest for multiple resistance. The choice of the specific product within a mode of actionwas mostly made following general recommendations at the Danish decision supportsystem for farmers plantevaern-online, 2014. An exception is the herbicidePursuit, which is not authorised on the Danish market, but adds another chemicalgroup of ALS inhibitors to the trial.

The following four ALS inhibitors (HRAC group B) were used (Hancock et al.,2007):

Primus with the active ingredient florasulam, N-(2,6-difluorophenyl)-8-fluoro-5-methoxy(1,2,4) triazolo(1,5-c)pyrimidine-2-sulfonamide, which is a Triazolopy-rimidine (TP) herbicide. It is a broadleaf herbicide. It is translocated bothin the xylem and phloem. The recommended dose of Primus for control ofCAPBP at 3-4 leaf stage in Denmark is 0.69 g ai/ha in spring barley at growthstage 22 (two tillers detectable) (plantevaern-online, 2014).

Lexus contains the active ingredient flupyrsulfuron- methyl-sodium, sodium saltof methyl 2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-6-(trifluromethyl)-3-pyridinecarboxylate. It belongs to the sulfonylurea (SU)group, more specific pyrimidinsulfonylurea. It is used for broadleaf and grassweed control and translocated in phloem, as well as xylem. The recommendeddose of Lexus for control of CAPBP at 3-4 leaf stage in Denmark is 0.3 g ai/hain winter wheat at growth stage 11 (first leaf unfolded) (plantevaern-online,2014).

Hussar OD contains the active ingredient iodosulfuron, 4-iodo-2[[[[4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl] benzoic acid andbelongs to the sulfonylurea, more specific triazinylsulfonylurea group. It istranslocated primarily in the phloem and applied for both broadleaf and grassweeds. The recommended dose of Hussar ODfor control of CAPBP at 3-4 leafstage in Denmark is 0.4 g ai/ha in spring barley at growth stage 22 (two tillersdetectable) (plantevaern-online, 2014).

Pursuit with the active ingredient imazapyr, 2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid, belongs

3.2 Herbicides 13

to the imidazolinones (IMI). It is used for both broadleaf and grass weedcontrol. It is translocated both in the xylem and phloem. Pursuit is not au-thorised in Denmark. Therefore no recommended dose is given for this herbicide.

These herbicides are all used for post emergence weed control. Treated plants showsymptoms like chlorosis, necrosis and plant death within two to three weeks afterapplication. With this choice of ALS inhibiting herbicides, three of the five existingclasses are covered.

The remaining herbicides have different modes of action (Hancock et al., 2007):

Starane 180S contains the active ingredient fluroxypyr, [(4-amino-3,5-dichloro-6-fluoro-pyridinyl)oxy]acetic acid, and belongs to the HRAC group O. Its chemicalstructure is similar to the auxin indole acetic acid. When present in the plantat high concentrations, plant cell growth and nitrogen metabolisation are dis-rupted, leading to leaf curling and premature senescence. The recommendeddose of Starane 180S for control of CAPBP at 3-4 leaf stage in Denmarkis 109.8 g ai/ha in spring barley at growth stage 22 (two tillers detectable)(plantevaern-online, 2014).

Glyfonova Plus contains the active ingredient glyphosate, N-(phosphonomethyl)glycine which belongs to the HRAC group G. Its mode of action is inhibitionof the enolpyrovyl shikimate-3-phosphate synthase, leading to depletion of thearomatic acids tryptophan, tyrosine and phenylalanine. It is a non selectiveherbicide, active on all plant species. Typical symptoms are growth inhibition,followed by chlorosis and necrosis which occur first in immature leaves and atgrowing points. Glyfonova Plus is not recommended for control of CAPBP inDenmark. Therefore no recommended dose is given.

Fighter with the active ingredient bentazone, 3-(1-methylethyl)- 1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide, belongs to the HRAC group C3. Itinhibits photosynthesis at photosystem II. Typical symptoms are chlorosis,followed by foliar desiccation and necrosis. Fighter is not recommended forcontrol CAPBP in Denmark. Therefore no recommended dose is given.

Stomp CS contains the active ingredient pendimethalin, N-(1-ethylpropyl)-3,4-dimethyl-2,6 dinitrobenzenamine, and belongs to the HRAC group K1. Itsmode of action is the inhibition of the microtubule protein tubulin, leading tomitotic disruption. As it is a typical pre emergence herbicide, highly susceptiblespecies fail to emerge. Other symptoms are brittle stems at the soil line androot growth inhibition. The recommended dose of Stomp CS for control ofCAPBP at 0-2 leaf stage in Denmark is 308 g ai/ha in spring barley at growth


stage 11 (first leaf unfolded) (plantevaern-online, 2014).

All product names will from now on only be referred to by the trade name withoutappendixes.

3.3 Characterisation experiment3.3.1 Pot experimentIn these experiments all CAPBP biotypes were followed during their whole growthperiod. The characterisation experiment was repeated three times with identicalexperimental set up. Two characterisation experiments (experiment 1 and 2) wereconducted in spring and one (experiment 3) in autumn with the sowing dates 12.02.14,25.02.14 and 07.08.14, respectively.

Table 3.2: Germination rate of the first experiment and experimental setup of all char-acterisation experiments. Experiment 1 ant 2 were conducted in spring and experiment3 in autumn. Displayed are the number of pots and the number of seeds per pot.

Biotype Germination (Exp. 1) Exp. 1 Exp. 2 Exp. 3

Pots Seeds Pots Seeds Pots SeedsID140 8.8 % 25 10 30 50 30 100ID1117 32 % 25 10 25 100 30 20ID1118 2.4 % 25 10 30 10 30 200ID1089 7.2 % 25 10 30 8 30 100ID1120 29 % 25 10 25 50 30 20ID1123 49.6 % 25 10 25 6 30 20

For all three experiments, seeds were sown in two litre pots according to table 3.2,covered with gravel and irrigated to field capacity. Due to a low germination of lessthan 10 % for most of the susceptible biotypes (table 3.2, Exp. 1), the amount of potsand seeds per pot changed over the experiments. The reason for this was a limitedavailability of seeds, especially for ID1120, ID1123 and ID1117. Once the plantsgerminated, they were thinned to one plant per pot. The further analysis was carriedout on a total of 20 plants per biotype. These plants were followed during their earlygrowth and the time to two and four leaf stage, time to the appearance of floweringprimordium and time to flowering were recorded. At the appearance of floweringprimordia 5 plants per biotype were harvested to measure rosette diameter, leaf areaand leaf number. Rosette diameter was measured in two diagonal directions of whichthe mean was taken to compensate for uneven shapes of the rosette. For measuringleaf area the leaf area meter Li-core (model 3100) was used. At the time of floweringanother five plants per biotype were harvested and the same measurements wereundertaken. Once the seeds were ripe, another harvest, including the measurement

3.4 Dose-response experiment 15

of flower stem height and separate measurements of leaf- and inflorescence fresh-and dry weight, was carried out. The inflorescences of the remaining five plants perbiotype were dried at room temperature to collect seeds. For each plant, thousandseed weight was determined individually. Subsequently, the seed samples were usedto conduct germination experiments.

3.3.2 Germination experimentsAll germination experiments were conducted in 9 cm petri dishes with two layers offilter paper (AGF 118-85 mm Frisenette Aps). For all four replicates each petri dishwas prepared with 50 seeds.

One germination experiment was designed to find the best germination conditions forCAPBP. Seeds were germinated at three different temperatures: at 25 ∘C, 17 ∘C orat alternating 10/17 ∘C (10h 10 ∘C and 14h 17 ∘C) in darkness. At each temperaturegermination at five different concentrations of KNO3 was tested. At the start of theexperiment 2.5 ml of one of the following solutions was added to each petri dish:0 g/l, 0.5 g/l, 1 g/l, 2 g/l and 10 g/l KNO3. The seed material used was seeds fromthe original samples for ID140 and ID1117 and progeny of ID1120 and ID1123. Thisprogeny of ID1120 and ID1123 derives from plants grown in separate isolation tentsto exclude the possibility of outcrossing with pollen from other biotypes. Brieflythis experiment contains 3 (temp.) X 5 (concentrations KNO3) X 4 (biotypes) X 4(replicates) = 240 petri dishes.

The germination experiment with progeny of plants of the characterisation experi-ments was conducted with seeds from five individuals per biotype. In this experiment1 g/l KNO3 solution was added to each petri dish and it was conducted at 25∘C.This means, it contains 5 (individuals) X 6 (biotypes) X 1 (concentration KNO3) X4 (replicates) = 120 petri dishes.

3.4 Dose-response experimentThe dose-response experiment was conducted with four different CAPBP biotypes.Two resistant, ID1120 and ID1123, and two susceptible, ID140 and ID1117. Thechoice on doses and dilution factors of the herbicides was based on the results of apilot test with ID1117 (results not shown).

3.4.1 Post emergence herbicidesThe experimental setup for spraying with post emergence herbicides (POST) was acompletely randomised plot design in a sheltered outdoor area with two litre pots,filled with a potting mixture. This potting mixture consists of peat, sand and sandyloam in a proportion of 2:1:2 w/w, respectively. Before sowing all seeds were imbibedin 1 g/l KNO3 solution for approximately three to six hours. Subsequent, seedswere sown in trays filled with the same soil, covered with gravel for germination and


irrigated to field capacity. At the four to six leaf stage plants were transplanted withthree plants in each pot. Four days after transplanting the plants the pots weresprayed in a spraying cabinet with a two nozzle boom and a pressure of 3 bar, a speedof 5.4 km/h and a spraying volume of 159 l/ha. The exact doses are shown in table3.3 in the appendix. The maximum doses applied on the susceptible and resistant (inparenthesis) biotype, corresponds to 0.57 (2.3), 2 (32), 2 (32) times the recommendeddose sprayed with Primus, Lexus and Hussar, respectively (plantevaern-online,2014). Due to the fact that Pursuit is not authorised in Denmark, no recommendeddose is given for this herbicide. The herbicides with other modes of action wereapplied at equal doses to all biotypes. The highest dose for Starane was 1.21 times therecommended dose. Neither Glyphonova, nor Fighter are recommended herbicidesfor the control of CAPBP and therefor no recommended dose is given.

All four replicates were sprayed with seven doses, plus control. All biotypes re-ceiving the same dose were sprayed simultaneously but every replicate was sprayedseparately, ensuring true replication. The experiment was harvested 28 days afterspraying. Fresh- and dry weight per pot were collected.

3.4.2 Pre emergence herbicideThe experiment with the pre emergence herbicide (PRE) Stomp was also a completelyrandomised block design, located in a sheltered outdoor area. Here, one litre potsfilled with field soil were used. The soil was sandy loam, collected at a field at theresearch station Flakkebjerg. Before sowing the seeds were imbibed in 1 g/l KNO3solution for approximately three hours. Exactly 20 seeds per pot were sown, coveredwith gravel and irrigated until field capacity was reached. The pots were sprayed theday after sowing using the same method, as presented for the POST experiment. Theminimum and maximum doses are equal to 0.01 and 0.47 times the recommendeddose, respectively. After spraying the pots were placed on individual saucers andirrigated with 5 mm water directly onto the top soil layer, two and 24 hours afterspraying. The experiment was harvested seven weeks after spraying. At harvestthe number of plants per pot were counted and fresh- and dry weight per pot weremeasured.

3.4 Dose-response experiment 17

Tab

le3.

3:H

erbi

cide

san

ddo

ses

(gai

/ha)

for

the

susc

eptib

le(I

D14

0an

dID

1117

)an

dre

sista

nt(I

D11

20an

dID

1123

)bi

otyp

es.

Ssu

scep

tible

,Rre

sist

ant Pr

imus

Lexu

sH

ussa

rPu

rsui

tSt

aran

e18

0SG

lyfo

nova

Plus

Figh

ter

Stom

pS

RS

RS

RS

RS

+R

S+

RS

+R

S+

RD

ilutio

nfa

ctor

22

22

22

22

31.

63

21

0.00

625

0.03

10.

0125

0.25

0.19

78.

583

0.65

825

20.

0125

0.06

20.

025

0.5

0.59

213

.73

1.97

550

30.

025

0.02

50.

125

0.05

11.

778

21.9

735.

926

100

40.

050.

050.

250.

250.

10.

12

25.

333

35.1

5617

.778

200

50.

10.

10.

50.

50.

20.

24

416

56.2

553

.333

400

60.

20.

21

10.

40.

48

848

9016

080

07

0.4

0.4

22

0.8

0.8

1616

144

144

480

80.

84

1.6

329

1.6

83.

264

1032

12.8

256


3.5 Competition experimentThe competition experiment was conducted as a target- neighbourhood experimentas described in Walsh et al., 2008. The CAPBP plant is the target, surrounded byneighbouring spring barley (SB) plants at different densities (figure 3.1).

Five litre pots were filled with the same potting mixture as in the characterisa-tion experiment (section 3.3). Seeds of spring barley (SB) (variety Evergreen) weresown at 2.5 centimetre depth using a master templates to ensure similar distributionof plants in all pots. The setup consisted of five densities: 0, 2, 4, 8 and 16 SB plantsper pot. This corresponds to 0, 53, 105, 210 and 421 plants per m2, respectively (seefigure 3.1). Each density was replicated four times, except for the pots without SB,which were replicated 12 times.

In addition to the pots 920 SB seeds were sown in trays as backup for non- germinatedSB plants in the setup. When needed the SB plants were replaced after germinationin the second week after the start of the experiment.

Fig. 3.1: Set-up of the competition experi-ment. In each pot there is a CAPBP plantplaced in the center and surrounded by 0, 2,4, 8 and 16 SB plants (from left to right).

CAPBP plants were sown separately intrays and transplanted at the four to sixleaf stage. According to the randomi-sation plan of a completely randomisedplot design one CAPBP plant of the fourbiotypes (ID140, ID1117, ID1120 andID1123) was placed in the center of eachfive litre plot. The whole experiment wasreplicated once. With an interval of oneweek, the experiments were placed at two opposite corners of the semi field area.

The first competition experiment was harvested after a growing period of 8 weeksand the second after 10 weeks. This difference in growing periods was due to diseasein the first experiment. Ramularia was identified on the leaves of SB 7 weeks afterstart of the experiment and it was therefore sprayed with 0.25% Comet and 0.25%Bell. Additionally, there was strong attack by insects (not identified), which ledto heavy injury of the plants of the first experiment. Consequently, both CAPBPand SB plants were in a deplorable condition and had to be harvested. The secondexperiment was not affected by sickness.

3.6 Genetic analysis3.6.1 DNA ExtractionFor extraction 100 mg plant material from ID1123 (R) and ID1117 (S) was collected.Each sample was filled in two ml microcentrifuge tubes and immediately frozen withliquid nitrogen. Subsequently, the samples were grinded at 1500 rps for 45 secondsand stored at -80∘ C. For the extraction the DNeasy Plant Mini Kit (50, cat. no.

3.6 Genetic analysis 19

69104; Qiagen) was used and the instructions in the manual were followed. The lastelution step was done in two steps with 100 𝜇l AE buffer and then a second timewith 50 𝜇l AE buffer, giving a total DNA volume of 150 𝜇l sample. The Nano DropSpectrophotometer was used to determine the concentration of extracted DNA inevery sample. All samples containing less than 100 ng/𝜇l DNA were discarded.

3.6.2 Polymerase chain reactionThe polymerase chain reaction (PCR) was conducted according to Cui et al., 2012.Their primer sequences two and four were used, here referred to as primer pair oneand two (table 3.4). These two primer pairs cover a DNA fragment of the DNAcoding for the ALS gene, containing seven out of the nine confirmed point mutationsfound in CAPBP Cui et al., 2012, table 3.4.The PCR was conducted in a totalvolume of 25 𝜇l according to the master mix in table 3.5. Four different preparationswere made. One with MgCL2 added and a low amount of DNA, one with MgCL2and a larger amount of DNA, one without MgCL2 and with a low amount of DNAand one without MgCL2 and a big amount of DNA.

Table 3.4: Primer sequences used for the amplification of two ALS gene fragments.The sequences were taken from Cui et al., 2012.

Primer Sequence (5’-3’) Amplicon size (bp) Point mutations contained

Forward 1 CAAGGAGGTGTATTCGCAGC 783 Pro197, Ala205, Asp376, Arg377Reverse 1 CTTATTCTTCCCAATCTCAGCCGForward 2 AAGGGATGAACAAGGTGCTT 765 Trp574, Ser653, Gly654Reverse 2 TGTCTCTCAGTATTTCGTCCG

The PCR cycles were set to a 3 min denaturation at 94 ∘C; 25 cycles of 30 s at 94∘C,30 s at 60∘C (primer 1) or 57∘C (primer 2), and 1 min at 72∘C; then 3 min at 72∘C(Cui et al., 2012).

3.6.3 Gel electrophoresisThe gel elecrophoresis was conducted in 1.5 % agarose gel (7.5 g ultra pure agarosein 500ml 1x TAE buffer). For DNA staining 10𝜇𝑙/100 𝑚𝑙 𝑔𝑒𝑙 SYBR safe DNA gel stain(Invitrogen) was used. After the gel was cooled down, it was loaded with 5 𝜇l of 1xloading dye and 5 𝜇l PCR product for primer 1 and 1 𝜇l 6x loading dye and 5 𝜇lPCR product for primer 2. The volume differs because the pockets were too smallto hold a total volume of 10 𝜇l. As reference for the size of the PCR product 10 𝜇l


GeneRuler exACTGene 100bp DNA Ladder was placed next to the samples. The gelran for 60 min at 110 V and 400 mA.

The gel was visualised using the Gel logic 100 image system and the Pharmacia LKBMacroVue. All PCR products were with a size of 850 bp ca. 70 bp longer thanexpected. In spite of that one pcr product per primer pair and biotype was sent forsequencing to MacroGen.

The chromatograms were checked by eye and the irregular ends were cut usingthe BioEdit sequence alignment editor (Hall, 1999). Subsequently, the forward andreverse (inverted) sequencing result of primer pair one were aligned together with thefully sequenced ALS gene (Jin et al., 2011) (genbank accession number HQ880661.1)and a defined sequence giving the start of the codon of amino acid 191 (from Cuiet al., 2012). This starting point was used to identify possible point mutations in therange of primer pair one (table 3.4). The signal of primer pair two was too weak foranalysis and was therefore discarded.

Table 3.5: Overview of the ingredients for a 1x master mix in 𝜇l, used for PCR.

+ MG - DNA + MG + DNA - MG - DNA - MG + DNA

H2O 16 15.25 20 19.5Buffer (5x) 5 5 2.5 2.5dNTP (10 mM) 0.5 0.5 0.5 0.5MgCl2 (25 mM) 1.5 1.5 - -Primer F 0.75 0.75 0.75 0.75Primer R 0.75 0.75 0.75 0.75Taq (5 𝑢/𝜇𝑙) 0.25 0.25 0.25 0.25DNA 0.25 0.75 0.25 0.75Total volume 25 25 25 25

3.7 Statistical analysis 21

3.7 Statistical analysisAll the ecological data was analysed with the statistical program R version 3.0.3(R-CoreTeam, 2014).

CharacterisationThe main objective of the characterisation experiment was to identify if biotypesdiffered in a number of parameters. In order to do so, an analysis of variance(ANOVA) model was fitted with the aov() function in R. The data was checked forthe statistical assumptions of normal distribution of residuals and homoscedasticity.When necessary the data was transformed.

Subsequently, if there was a significant difference between biotypes and the nullhypothesis H0 could be rejected, pairwise least squares means for the six differentbiotypes were calculated with the function lsmeans(). Finally, the functions glht()and cld() were used to group biotypes according to their statistically significantdifferences. Similar biotypes were assigned the same letters and different letters meanthat the biotypes are statistically significant different from each other. The resultsare shown in the form of boxplots in section 4.1.

Dose-response curvesThe dose response curves were analysed with the package drc according to thedescription in the user manual Ritz and Streibig, 2012. At first, the models werefitted according to the four-parameter logistic function (Ritz and Streibig, 2005),given by the formula:

𝑓(𝑥,(𝑏,𝑐,𝑑,𝑒)) = 𝑐 + 𝑑 − 𝑐

1 + 𝑒𝑥𝑝{𝑏(𝑙𝑜𝑔(𝑥) − 𝑙𝑜𝑔(𝑒))} (3.1)

whereas d and c are the upper and lower limits of the curve. The parameter e isthe point where half the investigated effect is measured, and b is the slope arounde. When interpreting e, it can be defined as effective dose (ED) where biomass isreduced by 50%. This is abbreviated with ED50. For a graphical explanation, seefigure 3.2.

In order to fulfil the assumption of normally distributed data, a BoxCox powertransformation was applied to data from each herbicide separately. Since the lowerlimit c for any of the herbicides did not differ significantly from 0, it was possible toreduce the model to a three- parameter logistic function. The resulting function hasthe form:

𝑓(𝑥,(𝑏,𝑑,𝑒)) = 𝑑

1 + 𝑒𝑥𝑝{𝑏(𝑙𝑜𝑔(𝑥) − 𝑙𝑜𝑔(𝑒))} (3.2)

Also for this three-parameter model, a BoxCox power transformation was applied, if


necessary.

Example for a dose−response curve

0 0.01 0.1

0

20

40

60

80

100

120

Dose (g ai/ha)

Rel

ativ

e fr

eshw

eigh

t

d

e, b

c

Fig. 3.2: Example of a curve of a four- pa-rameter logistic model. It shows the upperlimit d, the location of the ED50 e, the slopearound e, described by the parameter b andthe lower limit c. Note that the x- axis isdisplayed on a log scale and broken to havea correct representation of zero.The curverepresents the results of Primus on ID140.

The ED50 values and other statisticalparameters that can be found in tableswere calculated on the basis of dry weightdata. For the figures, relative freshweight was taken to obtain smoothercurves.

The resistance index (RI) was calculatedby dividing the ED50 of the resistantbiotype by the ED50 of the susceptiblebiotype. It thereby is an easily inter-pretable indicator of resistance. WhenRI=1, both biotypes are equally sensitiveto the herbicide. When RI>1, there isevidence for resistance and when RI<1,the resistant biotype is more sensitive tothe herbicide, than the susceptible bio-type.

Germination experimentsFor analysis of the germination experiment, the cumulative distribution function forthe standard three parameter log- logistic distribution (equation 3.2) was used (Ritz,Pipper, et al., 2013):

𝐹 (𝑡) = 𝑑

1 + 𝑒𝑥𝑝[𝑏{𝑙𝑜𝑔(𝑡50)}] = 𝑑

1 + ( 𝑡𝑡50

)𝑏(3.3)

Where the upper limit d is the proportion of seeds germinating during the ex-periment and t50 is the timepoint when 50% of the seeds germinating during theexperiment have germinated. This timepoint has the same interpretation as the ED50in the log-logistic model (equation 3.2). At t50 the slope of the curve is described bythe parameter b.

Competition experimentThe analysis of differences between densities and between biotypes was conducted inthe same way, as the statistical analysis of the characterisation experiment (section 3.7).The model on CAPBP dry weight as a function of SB plant density was fitted accordingto a four parameter log- logistic model with the package drc.

CHAPTER 4Results

4.1 Characterisation experiment4.1.1 Pot experimentCorrelogramThe two correlograms in figure 4.1 show correlations between traits of the resistantand susceptible biotypes of experiment 2. With some exceptions, it was just thestrength of correlations that differed between the correlogram on the resistant andthe correlogram on susceptible biotypes. One of theses exceptions was the negativecorrelation between time to germination/ time to four leaf stage and fresh- and dryweight of leaves at rosette stage (figure 4.1(a)). This correlation was positive forsusceptible biotypes (figure 4.1(b)). Further, there were some differences when itcame to the correlation between parameters representing reproductive and vegetativegrowth. Whereas the correlations of fresh- and dry weight of the inflorescence and leafarea, leaf number or rosette diameter was mostly negative for the resistant biotypes,it was positive for the susceptible biotypes. Further, the correlation of flower heightand fresh- and dry weight of leaves at the end of the growth phase was negative forresistant and positive for susceptible biotypes.

Phenological traitsAutumn sown CAPBP plants had a faster plant development until the appearanceof flowering primordium, compared to spring sown CAPBP plants (figure 4.2 (a) to(d)). The same relationship held for plant development until flowering, but therewas no longer a significant difference in days to flowering of spring and autumn sownCAPBP plants of ID1118, ID1120 and ID1123. In contrast to this fast developmentof autumn sown CAPBP plants, compared to spring sown plants, was the number ofdays from the occurrence of the flowering primordium to flowering. Here, autumnsown plants were slower in their development, compared to spring sown plants. Eventhough this trend accounted for all biotypes, it was only significant for ID140 andID1123.

Despite the differences in the length of CAPBP spring and autumn life cycles,

23

24 4 Results

germ

2leaf

4leaf

prim

flow

hight

rosette

LN

LA

SN

TSW

Rfresh

Rdry

Ffresh

Fdry

Hfresh

Hdry

Infl.fresh

Infl.dry

(a) Correlogram of the resistant biotypes, ID1120 and ID123

germ

2leaf

4leaf

prim

flow

hight

rosette

LN

LA

SN

TSW

Rfresh

Rdry

Ffresh

Fdry

Hfresh

Hdry

Infl.fresh

Infl.dry

(b) Correlogram of the susceptible biotypes, ID140, ID1117, ID1118 and ID1089.

Figure 4.1: Correlograms of characterisation experiment two. They show the correlationbetween specific phenological and morphological traits of CAPBP. The upper part ofthe figure shows the correlation between two traits as dots in a plot. In the lower panelstrong correlations are represented by intensive colours, weak by pale colours. Redmeans negative correlation and blue positive. (germ= days to germination; 2leaf= days to twoleaf stage; 4leaf= days to four leaf stage; prim= days to flowering primordium; flow= days to flowering;height= flower height; rosette= rosette diameter; LN= leaf number; LA= leaf area; SN= seed number;TSW= thousand seed weight; Rfresh/ Rdry= fresh-/ dry weight at rosette stage; Ffresh/Fdry= fresh-/drywight at flowering; Hfresh/Hdry= fresh-/dry weight at harvest ; infl.fresh/infl.dry= fresh/dry weightof the inflorescence.)

4.1 Characterisation experiment 25

68

1012

1416

18

Day

s to

ger

min

atio

n

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

c

a

b

a

d

a

bc

a

bc

a

b

a

(a) Days to germination

1015

20

Day

s to

2 le

af s

tage

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

c

a

b

a

c

a

c

a

bc

a

bc

a

(b) Days to two leaf stage

1520

25

Day

s to

4 le

af s

tage

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

bc

a

b

a

c

a

bc

a

c

a

bc

a

(c) Days to four leaf stage

2030

4050

6070

80

Day

s to

flow

erin

g pr

imor

dium

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

ef

cd

e

bc

def

ab

de

a

fe

f ef

(d) Days to flowering primordium

3040

5060

7080

90

Day

s to

flow

erin

g

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

d

bc

ce

b

abcd

ab

bc

a

dcd

d de

(e) Days to flowering

24

68

10

Day

s fr

om fl

ower

ing

prim

ordi

um to

flow

erin

g

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

ad

e

ab

ad

acd

ce

a

ab

ad

bcd

ab

de

(f) Days from flowering primordium to flow-ering

Figure 4.2: Boxplots on the duration from sowing to specific growth stages and fromflowering primordium to flowering. For each biotype, the results of the spring sown(blue) and autumn sown (white) experiments are shown separately. Biotypes markedwith different letters are significantly different from each other.

26 4 Results

life cycle patterns of spring sown plants resembled those of autumn sown plants. Atboth times of the year, all biotypes had a similar development in the early growthstages, up to four leaf stage. However, when it came to the production of floweringprimordia and flowering, it was observable that the resistant biotypes, ID1120 andID1123, initiated the reproductive growth phase at a later time point, compared tomost of the susceptible biotypes. This relationship was not always significant forID140, which was also flowering relatively late.

Morphological traitsThe boxplots of plant dry weight at different development stadia (figure 4.3) show,that ID140, ID1120 and ID1123, had a significantly higher dry weight at rosettestage, compared to the remaining biotypes. At flowering, the dry weight of ID1089was very low, compared to the other biotypes. Apart from this, biomass at floweringwas quite homogeneous. At the end of the growth phase, the resistant biotypes hada tendency to higher dry weights, compared to the susceptible biotypes. In contrastto this, the dry weight of the inflorescence tended to be lower for resistant, comparedto susceptible biotypes. This was most explicit for ID140, compared to ID1120 andID1123. However, these differences were not significant.There was no pronounced difference in dry weight between spring- and autumn sownCAPBP plants.

Figure 4.4 shows rosette diameter, leaf area (LA) and leaf number (LN) at flowering.This development stage was chosen because there had been an irrigation breakdown,that led to drought stress of ID140, ID1120 and ID1123 between flowering andthe end of the growth phase of experiment three. Therefore, results of these bio-types recorded after flowering might not be representative. Results of LA and LN atrosette stage and at the end of the growth phase are shown in the appendix, figure A.5.

The boxplots of rosette diameter, LA and LN at flowering and flower height atthe end of the growth phase all show a homogeneous pattern with no significantdifferences between years or biotypes.

Figure 4.5 shows thousand seed weight (TSW) and seed number (SN) per plant ofthe different CAPBP biotypes. There was no significant difference between biotypesof either seed characteristic. However, the pattern of the boxes shows a larger TSWof ID140, compared to the other biotypes. Further on, ID1117 had a tendency to ahigher amount of seeds per plant, compared to the other biotypes.

In summary, ID140, ID1120 and ID1123 were the most vigorous biotypes, producedmost biomass and ID1089 was the biotype that produced the least biomass. Therewas neither difference between resistant and susceptible biotypes in the parametersmeasured concerning plant morphology, nor phenology.


01

23

4

Dry

wei

ght a

t ros

ette

sta

dium

(g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

cd

d

ab

aa a

bcbc

cd

c

(a) Dry matter at rosette stage

05

1015

Dry

wei

ght a

t flo

wer

ing

(g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

ce

de

bc

be

bc

bcd

a b

ce

ce

ce

e

(b) Dry matter at flowering.

05

1015

2025

Dry

wei

ght a

t the

end

of t

he g

row

th s

tage

(g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

dd

bc

d

cd

ca ab

d

d

d

d

(c) Dry matter of the leaves at the end ofthe growth phase.

010

2030

40

Dry

wei

ght o

f the

inflo

resc

ence

(g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

ac bcc

acac

ac

a

aca

aba

(d) Dry matter of the inflorescence at theend of the growth phase.

Figure 4.3: Boxplots on plant dry weight of different CAPBP biotypes at differentdevelopment stadia. For each biotype, the results of the spring sown (blue) and autumnsown (white) experiments are shown separately. Biotypes marked with different lettersare significantly different from each other.

Leaf typeAs described in section 2.1, the species CAPBP was very diverse and could be dividedin four different leaf types (Shull, 1909). Pictures of a representative individual ofeach biotype were presented in figure 4.6. It shows the whole plant at rosette stage, aswell as three leaves of different sizes. Hereby the smallest leaf was the most recentlyemerged, taken from the center of the rosette. The leaves of ID140 and ID1117 weredivided into the midrib and the terminal lobe was set off from the nearest lateral lobe.As the leaves of both biotypes did not show a rounded portion close to the midriband elongated portions at the tip, they were assigned to the leaf type rhomboidea.All remaining biotypes, ID1089, ID1118, ID1120 and ID1123 had leaves that werenot divided into the midrib. Whereas the ones of ID1089 and ID1118 were veryshallow on the edges, characteristic for leaf type simplex, ID1120 and ID1123 showedpronounced incisions and were therefore assigned leaf type tenuis.

28 4 Results

010

2030

4050

6070

Ros

ette

dia

met

er a

t flo

wer

ing

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

cdede

bc

be

a

b

be

bd

dede

cdee

(a) Rosette diameter at flowering.

050

015

0025

00

Leaf

are

a at

flow

erin

g (c

m)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

dfg

g

ac

bcf

acd

bcde

aab

dfg cg

efg

fg

(b) Leaf area at flowering.

050

100

150

200

250

Leaf

num

ber

at fl

ower

ing

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

effg

bc

ce

bcde

c

aab

dfg

fg

fg

g

(c) Leaf number at flowering

2040

6080

100

120

Flo

wer

hig

ht (

cm)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

dbd

d bd

abc

abc

a

d

ab

cd

ab

(d) Flower height at the end of the growthphase.

Figure 4.4: Boxplots on morphological traits of CAPBP biotypes. For each biotype,the results of the spring sown (blue) and autumn sown (white) experiments are shownseparately. Biotypes marked with different letters are significantly different from eachother.

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

0.00

0.04

0.08

0.12

Tho

usan

d se

ed w

eigh

t (g)

b

aba

abab ab

(a) Thousand seed weight

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

040

000

8000

012

0000

See

d nu

mbe

r pe

r pl

ant

a

a

aa

aa

(b) Seed number

Figure 4.5: Boxplots on seed characteristics of different CAPBP biotypes. The seedsoriginate from spring sown plants. Biotypes marked with different letters are significantlydifferent from each other.


Figure 4.6: Leaf types of six CAPBP biotypes. From left to right it is displayedID140 (R), ID1117 (R), ID1118 (S), ID1089 (S), ID1120 (T) and ID1123 (T)R= rhomboidea, S= simplex T= tenuis.

4.1.2 Germination experimentsGermination conditionsFigure 4.7 shows two resistant CAPBP biotypes, ID1120 and ID1123 and two suscep-tible CAPBP biotypes, ID140 and ID1117, germinated at three different temperatures(25 ∘C, 17 ∘C and alternating 10/17 ∘C) in five different concentrations of potassiumnitrate in darkness. The seeds used for this experiment were seeds from the originalseed samples for ID140 and ID1117 and progeny of ID1120 and ID1123.

At all temperature conditions treatments with potassium nitrate showed a bet-ter germination, than treatments with water. For the susceptible biotypes, treatmentwith 2 g/l KNO3 showed the highest germination. However, for the resistant biotypesit was 10 g/l KNO3 at 20 ∘C and again 2 g/l KNO3 at lower temperatures that hadthe best effect on total germination.

The highest percentage of germinated seeds was at high temperatures for susceptibleand at low, alternating temperatures for resistant biotypes. Table 4.1 show estimatednumbers of the days to 50% germination and total % germinated of the treatmentwith 2 g/l KNO3. The comparison of resistant with susceptible biotypes in table4.2 shows a clear difference between warm and cold temperatures. At 25 ∘C, totalgermination was always significantly higher for ID140 and ID1117 than ID1120 andID1123. This relationship was the complete opposite at low temperatures (figure4.2).

Further on, germination generally started earlier at high, compared to low tem-peratures (figure 4.7 and table 4.1). When comparing germination rates of susceptibleand resistant biotypes, ID1123 always had a slower germination rate, independent ontemperature (table 4.2). ID1120 germinated also slower than ID1117, but ID1120germinated faster than ID140 at 25 ∘C, though the latter was not significant (table4.2).

30 4 Results

It was interesting to see, that both resistant biotypes had two flushes of germi-nation at 25 ∘C, whereas both susceptible biotypes did not show this pattern. ID1120also showed this pattern at alternating 10/17 ∘C, though not as pronounced as theresistant biotypes (figure 4.7).

Table 4.1: Number of days to 50% germination of five CAPBP biotypes at 25 ∘C,alternating 10/17 ∘C and a concentration of 2 g/l KNO3. Each value is given togetherwith its standard error.

Days until 50% germination Total percentage germinated25 ∘C 10/17 ∘C 25 ∘C 10/17 ∘C

ID140 2.39 ± 0.079 12.83 ± 0.173 96.27 ± 0.033 87.87 ± 0.024ID1117 1.16 ± 0.033 5.34 ± 0.202 91.67 ± 0.020 79.17 ± 0.028ID1120 2.12 ± 0.156 14.87 ± 0.217 7.50 ± 0.019 100.00 ± 0.013ID1123 3.42 ± 0.367 12.95 ± 0.228 58.00 ± 0.038 100.00 ± 0.012

Table 4.2: Comparison of resistant and susceptible biotypes at 25 ∘C, alternating10/17 ∘C and a concentration of 2 g/l KNO3. Compared are the parameters days to50% germination and total percentage germinated. The fraction of the resistant dividedby the susceptible biotype is given together with the standard error. All parameterssignificantly different from 1 are marked with an asterix (p< 0.05).

Ratio of Ratio ofdays until 50% germination total germination (in %)

25 ∘C 10/17 ∘C 25 ∘C 10/17 ∘C

ID1120/ID140 0.89 ± 0.072 1.16* ± 0.023 0.07* ± 0 1.13* ± 0.000ID1120/ID1117 1.82* ± 0.143 2.78* ± 0.113 0.08* ± 0 1.26* ± 0.000ID1123/ID140 1.43* ± 0.161 1.00 ± 0.022 0.60* ± 0 1.14* ± 0.001ID1123/ID1117 2.93* ± 0.326 2.42* ± 0.101 0.63* ± 0 1.26* ± 0.001


ID140 at 25 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20

0 g/l KNO30,5 g/l KNO31 g/l KNO32 g/l KNO310 g/l KNO3

Days after start of the experiment

Per

cent

ger

min

ated

ID140 at 17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20

Days after start of the experimentP

erce

nt g

erm

inat

ed

ID140 at 10/17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1117 at 25 °C

0.0

0.2

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0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1117 at 17 °C

0.0

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0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1117 at 10/17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20

Days after start of the experimentP

erce

nt g

erm

inat

ed

ID1120 at 25 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1120 at 17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1120 at 10/17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1123 at 25 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1123 at 17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

ID1123 at 10/17 °C

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 20


Per

cent

ger

min

ated

Figure 4.7: Overview of all germination curves of four CAPBP biotypes at threedifferent temperatures and different concentrations of potassium nitrate.black/ circle= 0 g/l KNO3, red/ triangle= 0.5 g/l KNO3, green/ cross= 1 g/l KNO3, blue/ X= 2 g/l KNO3, turquoise/ quadrat= 10 g/l KNO3

32 4 Results

Comparison of germination of the CAPBP biotypes used in the characterisationexperimentThe seeds used in this experiment were offspring of the plants described in thecharacterisation experiment.ID1120 and ID140 were germinating slowest with 5.23 and 3.82 days to 50% germina-tion, respectively. All other biotypes lay in the range of 2.5 to 3 days to germination(table 4.3). In addition to this, table 4.4 showed that ID1120 always germinatedslower, compared to the susceptible biotypes. But ID1123 germinated with the samespeed or faster than the susceptible biotypes. The same relationship accounted forresistant and susceptible biotypes to 90% germination (table 4.4).

Total germination was highest for ID1123 and ID140 with 70% and 79% respec-tively. ID1120 and ID1118 had a similar total germinability of 40% and ID1089and ID1117 had the lowest total germinability of 36% and 25%, respectively (table 4.3).

It was noticeable, that the two biotypes with the highest total germination, ID140and ID1123, showed two flushes of germination, whereas all other biotypes did not(see figure 4.8).


Per

cent

ger

min

atio

n

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

2 4 8 20

ID1089ID1117ID1118ID1120ID1123ID140

Figure 4.8: Germination curves of four susceptible (ID140, ID1117, ID1118, ID1089)and two resistant (ID1120, ID1123) CAPBP biotypes. The seeds were germinated at25∘C and 1 g/l KNO3.

4.2 Dose- response analysis 33

Table 4.3: Number of days to 50% germination and the total percentage germinated offive CAPBP biotypes of the seeds from characterisation experiment two. Each value isgiven together with its standard error.

Days until 50% germination Total germination (in %)

ID1089 2.81 ± 0.251 25.33 ± 0.015ID1117 2.46 ± 0.069 36.63 ± 0.013ID1118 2.99 ± 0.100 39.36 ± 0.016ID1120 5.23 ± 0.269 40.38 ± 0.017ID1123 2.39 ± 0.070 70.24 ± 0.016ID140 3.82 ± 0.194 79.40 ± 0.018

Table 4.4: Comparison of the number of days to 50% germination between susceptibleand resistant biotypes of the seeds from characterisation experiment two. The fractionof the resistant divided by the susceptible biotype is given together with the standarderror. All parameters significantly different from 1 are marked with an asterix (p< 0.05).

Ratio of Ratio ofdays until 50% germination Days until 90% germination

ID1120/ ID1089 1.86* ± 0.191 1.26 ± 0.238ID1120/ ID1117 2.12* ± 0.040 2.80* ± 0.285ID1120/ ID1118 1.75* ± 0.107 2.26* ± 0.237ID1120/ ID140 1.37* ± 0.000 1.03 ± 0.131ID1123/ ID1089 0.85 ± 0.080 0.50* ± 0.087ID1123/ ID1117 0.97 ± 0.040 1.11 ± 0.075ID1123/ ID1118 0.80* ± 0.036 0.90 ± 0.064ID1123/ ID140 0.62* ± 0.037 0.41* ± 0.042

4.2 Dose- response analysisFigure 4.9 shows the modelled dose-response curves of the ALS inhibiting herbicides.Each curve represents one biotype, whereas ID140 and ID1117 were expected to besusceptible and ID1120 and ID1123 were expected to be resistant to ALS inhibitors.The ED50 values, estimated on the basis of dry weight measurements, can be foundin (table 4.5).For all herbicides, except Pursuit, the ED50 doses of ID1120 and ID1123 weresignificantly higher than for ID140 and ID1117 with ID1120 showing higher levels ofresistance, compared to ID1123.High levels of resistance to the herbicide Primus were observed for ID1120, wherethe treatment resulted in RI of 17.84 and 24.45 (ID140 and ID1117) (4.6). Eventhe highest dose of 1.6 g ai/ha, or 2.3 times recommended dose, was not sufficientto cause a reduction in relative fresh weight below 20%. When taking the ED50values into consideration, it was obvious that ID1120 had a very high ED50 value ata dose of 0.52 g ai/ha, compared to the other biotypes that had ED50 values below

34 4 Results

Primus

0 0.01 0.1 1

020406080

100120 ID140

ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Lexus

0 0.01 0.1 1 10

020406080

100120 ID140

ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Hussar

0 0.01 0.1 1 10

020406080

100120

ID140ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Pursuit

0 0.1 1 10 100

020406080

100120

ID140ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Figure 4.9: Dose- response curves with relative fresh weight of all ALS inhibitingherbicides used in this experiment: Primus (florasulam), Lexus (flupyrsulfuron), Hussar(iodosulfuron), Pursuit (imazethapyr).

0.1 g ai/ha (4.5). Also the dose- response curve shown in figure 4.9, starts bendingat high doses and has a more convex shape, compared to the s shaped curves of theother three biotypes. In contrast to this, RI of ID1123 of 1.86 and 2.55, relative toID140 an ID1117, were too low to give evidence for resistance (4.6). Although, whenlooking at the curves in figure 4.9, the shape of the curve of ID1123 is different fromthe two susceptible biotypes. It has a very flat slope around the ED50, proceedshigher and finishes somewhat higher than the curves of the two susceptible biotypes.This indicated a differing response of ID1123 to Primus, without proving resistance.

Table 4.5: ED50 of all ALS inhibiting herbicides investigated in g ai/ha. Each value isgiven together with its standard error.

Primus Lexus HussarOD Pursuit

ID140 0.03 ± 0.009 0.04 ± 0.009 0.02 ± 0.003 1.02 ± 0.231ID1117 0.02 ± 0.006 0.07 ± 0.009 0.05 ± 0.007 1.57 ± 0.388ID1120 0.52 ± 0.181 1.70 ± 0.617 0.27 ± 0.081 3.17 ± 0.755ID1123 0.05 ± 0.025 0.53 ± 0.202 0.40 ± 0.134 1.10 ± 0.369

There was evidence for resistance of ID1120 and ID1123 to the herbicide Lexus, thoughlevels of resistance differed. RIs of ID1120 were with 37.9 and 25.9 (ID140, ID1117)distinct and three times the ones of ID1123 (table 4.6). Nevertheless, the ED50s


of ID1123 were with 12 and 8 times the ED50 of ID140 and ID1117, respectively,still pronounced and gave clear evidence for resistance. Both dose- response curvesof ID1120 and ID1123 showed a rather flat slope, compared to the two susceptiblebiotypes. ID1123 proceeded lower than ID1120, which was in line with the previousdescription of RI and ED50 values.

Table 4.6: Resistance indices of treatment with ALS inhibiting herbicides. Each valueis given together with its standard error. All values significantly different from 1 aremarked with an asterix (p< 0.05).

Primus Lexus Hussar Pursuit

ID1120/ ID140 17.84 ± 8.168 37.91* ± 15.774 11.07* ± 3.742 3.09* ± 1.015D1123/ ID140 1.86 ± 1.030 11.84* ± 5.110 16.83* ± 6.078 1.08 ± 0.435ID1120/ID1117 24.45* ± 11.258 25.89* ± 10.047 5.35* ± 1.786 2.01 ± 0.589ID1123/ID1117 2.55 ± 1.417 8.09* ± 3.272 8.13* ± 2.906 0.70 ± 0.291

The curves of Hussar OD visualise a clear difference between curve progression ofthe susceptible and expected resistant biotypes (figure 4.9). The curves of ID1120and ID1123 start bending at much higher doses, compared to ID140 and ID1117.This was confirmed by the RIs, which were a bit higher for ID1123 in comparisonto ID1120 but lay above 5 and therefore clearly indicated resistance of ID1120 andID1123 to the active ingredient iodosulfuron (table 4.6).

There was evidence for very equal control of all biotypes by the herbicide Pur-suit. The resistance indices of all biotypes were quite similar and with the highestvalue of 3.1 for ID1120/ID140, they were too low to prove resistance (table 4.6). Alsothe curves in figure 4.9 were similar in shape and position.

Figure 4.10 shows the results of spraying with herbicides of other modes of action,than ALS inhibition.

Table 4.7: ED50 values of Starane, Glyfonova and Fighter in g ai/ha. Each value isgiven together with its standard error.

Starane Glyfonova Fighter

ID140 105.10 ± 25.829 22.96 ± 2.676 50.26 ± 8.378ID1117 302.87 ± 556.019 27.24 ± 5.294 15.04 ± 5.067ID1120 94.17 ± 52.343 38.72 ± 5.480 71.46 ± 23.557ID1123 25.20 ± 28.435 48.65 ± 10.611 12.25 ± 5.507

The treatment with Starane resulted in very low control of all CAPBP biotypesbecause the doses chosen for spraying were not high enough to kill the plants. This

36 4 Results

Starane

0 0.1 1 10 100

020406080

100120

ID140ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Glyfonova

0 10 100

020406080

100120 ID140

ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Fighter

0 1 10 100

020406080

100120 ID140

ID1117ID1120ID1123

Dose (g ai/ha)

Rel

ativ

e fr

esh

wei

ght

Figure 4.10: Dose- response curves with relative fresh weight of Starane (fluroxypyr),Glyfonova (glyphosate) and Fighter (bentazon). It is not possible to fit a curve to thedata of Stomp (pendimethalin).

Table 4.8: Resistance indices of treatment with Starane, Glyfonova and Fighter. Eachvalue is given together with its standard error. All values significantly different from 1are marked with an asterix (p< 0.05)

Starane Glyfonova Fighter

ID1120/ID140 0.89 ± 0.545 1.68* ± 0.310 1.42 ± 0.525ID1123/ID140 0.24* ± 0.277 2.11* ± 0.520 0.24* ± 0.117ID1120/ID1117 0.31 ± 0.596 1.42 ± 0.340 4.75 ± 2.239ID1123/ID1117 0.08* ± 0.179 1.78 ± 0.520 0.81 ± 0.457

can also be seen on the picture of representative plants taken before harvest, figureA.9(a) in the appendix. Because of this bad fit no further conclusions could be drawnfrom the modelling results and there was no evidence for resistance characteristics tothe active ingredient fluroxypyr.

Application of Glyfonova resulted in very good control of all biotypes. RIs were withthe maximum of 2.1 low and all curves had a beautiful s shaped course with similarED50 values (figure 4.10 and table 4.7, 4.8). Therefore, cross resistance to the activeingredient Glyphosate could be ruled out.

There was variable control of the four investigated biotypes by the herbicide Fighter


with dose-response curves of different shape (figure 4.10). Whereas ID140 and ID1120had a very shallow course, ID1117 and ID1123 showed steeper slopes which resultedin more pronounced s- shaped curves. This lead to a very high ED50 value of ID1120,compared to the other biotypes (table 4.7) and also a relatively high resistance indexof ID1120/ ID1117 of 4.75 (table 4.8). This could have indicated resistance of ID1120to the active ingredient bentazone, but this result was not significant. Also, thepicture taken just before harvest shows, that plants of ID1120 were dead at therecommended dose of 480 g ai/ha, arguing against resistance of ID1120 to bentazone(figure A.9(c)).

There is no dose response curve shown for the PRE herbicide Stomp, becausegrowth of the plants was generally bad. Therefore, it was not possible to fit a curveto the measured fresh and dry weight data. A picture of representative plants isshown in the appendix in figure A.9(d).

In summary, there was clear evidence for resistance of both, ID1120 and ID1123,to Lexus and Hussar, with a clearly lower resistance level of ID1123, compared toID1120. Moreover, ID1120 was resistant to Primus. Whenever resistance was present,dose- response curves showed a more shallow slope of resistant, compared to suscepti-ble biotypes. Further on, the here presented results showed a clear susceptibility ofboth, ID1120 and ID1123, to Pursuit.Additionally, cross resistance to Starane, Glyfonova and Fighter could be ruled out.

38 4 Results

4.3 Competition experimentFigure 4.11 shows representative CAPBP plants of the first competition experimentat harvest. It is obvious, that the CAPBP plants without SB (0 SB) were bigger thanCAPBP plants with any density of SB plants. However, in this experiment therewere no significant differences in growth of CAPBP between densities of SB plants(figure 4.12(a)) and biotypes (not shown).

Fig. 4.11: Harvest of the competition experiment.CAPBP plants of four different biotypes, ID140,ID1117, ID1120 and ID1123, are shown after grow-ing together with different densities of SB (0, 2, 4, 8and 16 SB plants per pot).

The second competition exper-iment revealed a more complexcorrelation between CAPBP dryweight and SB density. Here,CAPBP dry weight of differentSB densities could be grouped(figure 4.12(b)). This meantthat SB 0 was different fromSB 4, SB 8 and SB 16, SB 2 wasdifferent from SB 8 and SB 16and SB 4 was different from SB 8and SB 16. The resulting pat-tern indicated a strong negativecorrelation between SB densityand dry matter of CAPBP, andshowed increased competition ofSB at high densities. Similar tothe first competition experiment, there was no significant difference in growth ofCAPBP between biotypes (figure 4.13).

0

5

10

15

20

25

30

Number of barley plants per pot

Dry

wei

ght o

f CB

P (

g)

SB 0 SB 2 SB 4 SB 8 SB 16

a

b b b b

(a) First TN experiment.

010

2030

4050

Number of barley plants per pot

Dry

wei

ght o

f CB

P (

g)


a

ab

b

cc

(b) Second TN experiment.

Figure 4.12: Mean of the dry weight of the four target CAPBP biotypes at differentdensities of spring barley of the first and the second competition experiment. Densitiesmarked with different letters are significantly different from each other.

4.3 Competition experiment 39

010

2030

4050

Dry

wei

ght o

f CB

P (

g)


Figure 4.13: Dry weight of four different CAPBP biotypes at different densities ofspring barley (SB). Biotypes are presented with a gradient from light to dark coloursand correspond to ID140, ID1117, ID1120 and ID1123 from left to right.

The observation of this clear correlation between CAPBP dry weight and SB densitywas the determining factor for the attempt to model this relationship. Hence, afour parameter logarithmic curve was fitted to the data of the second competitionexperiment and the effect of density on CAPBP dry weight was modelled. Figure 4.14shows the relationship between density and CAPBP dry weight, with 50% reductionof dry weight at a density of 103 plants per m2, 80% reduction at 241 plants per m2

and 90% reduction at a density of 396 plants per m2 (table 4.9).

Table 4.9: Spring barley densities with 50, 80 and 90 percent of CAPBP biomassreduction. The values are calculated by modelling the data from the second competitionexperiment with a three parameter log- logistic function.

SB density Std. Error Lower Upper

50% 103.30 39.732 24.965 181.64580% 241.08 183.224 −120.186 602.34890% 395.78 397.665 −388.303 1179.864

40 4 Results

Density of SB plants

Dry

wei

ght o

f CA

PB

P (

g)

0

4

8

12

16

20

20 40 80 200 400

Figure 4.14: Estimated curve on CAPBP dry weight at different densities of springbarley (SB). The usual field density is around 200 plants per m2, giving a reduction ofmore than 50 %.

4.4 Genetic analysis 41

4.4 Genetic analysisFigure 4.15 shows amplified fragments of primer pair one. All amplicons had pro-nounced bonds with a size of ca. 850 bp, which was ca. 70 bp larger than theexpected size of 783 bp.

When visually examining the alignment of the sequencing result of the resistant andthe susceptible biotype, it looks like a good fit (figure 4.16). With a match of 98%also the rest of the sequences were very similar. When the corresponding codons ofthe point mutations Pro197, Ala205, Asp376 and Arg377, located in the range ofprimer pair one, were compared to the susceptible biotype and the full ALS genesequence, none of these point mutations could be found in the generated sequences.

I ID 1

123

IV ID

112

3V

ID 1

123

III ID

111

7

+ - Mar

ker

I ID 1

123

IV ID

112

3V

ID 1

123

III ID

111

7

+ -ca. 850 bp

Figure 4.15: Results of the PCR with primer pair one. All amplicons have a size of ca.850 bp.

Figure 4.16: Alignment of the sequencing results of Primer pair one. It is shownfrom top to bottom: Forward sequencing result of the amplicon of primer pair one,reverse sequencing result of the amplicon of primer one (inverted), part of the completesequencing of the ALS gene of CAPBP Jin et al., 2011 and a short piece of the ALS,starting at amino acid position 191.

CHAPTER 5Discussion

The results of the characterisation experiments reflect the diversity of CAPBP de-scribed in literature (Aksoy et al., 1998; Iannetta et al., 2007; Neuffer, 2011).This confirms a good choice of susceptible biotypes by making it possible to accountfor natural diversity in the species CAPBP when comparing them to the resistantbiotypes found in Denmark.

CharacterisationThe fact that the characterisation experiments were conducted in the greenhousemakes it difficult to compare them to studies on plants of natural populations orother greenhouse studies with deviating conditions. Nevertheless, the observed mor-phological and phenological characteristics of the six biotypes are in accordance withother descriptions of CAPBP. For example were rosette diameter and flower heightsomewhat larger than the standard described by Aksoy et al., 1998, but correspondto the results of Iannetta et al., 2007. Also the reproductive capacity (figure 4.5(b))lay in the range of 5,000 to 90,000 seeds per plant, similar to the investigation ofHurka and Haase, 1982.

The difference in strength of correlations between the resistant and susceptiblebiotypes (figure 4.1), could be explained by the difference of origin of the biotypes.The resistant biotypes originated from the same population, collected in one fieldand were therefore very similar to each other. In contrast to this, the susceptiblebiotypes originated from distant locations in Europe. Therefore, there was a higherchance for the susceptible biotypes to exhibit conflicting characteristics, leading toweak correlations between traits.

This difference of origin of the resistant and susceptible biotypes could also bethe explanation for the conflictive correlation of time to germination/ four leaf stageand fresh- and dry weight at rosette stage between resistant and susceptible biotypes.Early germinating susceptible biotypes, like ID1089 and ID1117 (figure 4.2(a)), alsohad low dry weight at rosette stage (figure 4.3(a)). Hence, early germination of

43

44 5 Discussion

susceptible biotypes correlated with low dry weight and late germination with bigdry weight, which resulted in a positive correlation. When analysing the correlationof this trait for the resistant biotypes, it was only the factor of plant developmentinfluencing the correlation because the biotypes were so similar. This means thatearly germinating plants had a longer growth period until rosette stage, thus morefresh- and dry matter, which resulted in a negative correlation. According to Haweset al., 2005, rosette diameter is an indicator of resource capturing ability of CAPBPbiotypes. He observed, that biotypes that are flowering late have a small rosettediameter due to poor ability of the biotype to capture resources. The present resultswere contradictory to this observation, as the correlation between days until floweringand rosette diameter were positive for both, resistant and susceptible biotypes (figure4.1).

The observed negative correlation for resistant biotypes of fresh and dry weightof the inflorescence with LA, LN and rosette diameter was possibly not based on plantcharacteristics, but due to a too early harvest of the resistant biotypes. Both, ID1120and ID1123 were flowering late (figure 4.2(e)) and this trait was positively correlatedwith rosette diameter, LN and LA (figure 4.1). Due to increasing sickness and necro-sis of the plants, most individuals of ID1120 and ID1123 were harvested before allflowers faded. This resulted in small inflorescences of plants with a big vegetative part.

The timepoint of harvest is therefore also the most probable explanation for the lowdry weight of the inflorescences of ID1120 and ID1123, compared to the susceptiblebiotypes in figure 4.3(d).

The difference in the amount of days needed for plant development in the springand autumn experiment, could be explained by the difference in day length betweenseasons in Scandinavia. As described in section 4.1.1, autumn sown plants werefaster in their development, compared to spring sown plants up to the occurrenceof flowering primordia. This relationship was reversed when autumn sown plantsreached flowering in late October. During this late phase of plant development, springsown plants had long days in the end of June and therefore had a faster developmentfrom flowering primordium to flowering.

Nevertheless, sowing dates of the experiments conducted here are not in accor-dance with the natural annual cycle of CAPBP. Even though CAPBP can germinatethroughout the year (Aksoy et al., 1998), under undisturbed conditions germinationpeaks in April (Baskin et al., 2004). This matches well with sowing time of springcrops like SB. Consequently, both susceptible and resistant biotypes are able to finisha life cycle in 90 days, in both winter and spring sown crops, before harvest in August.

Leaf typeLeaf type belongs to the most important traits when characterising CAPBP (Shull,1909). Three out of the four originally described leaf types simplex, rhomboidea,

45

tenuis and heteris are found to be genetically distinct (Iannetta et al., 2007), therebyverifying this approved method of sub classifying the species CAPBP. Nevertheless,it remains difficult to visually distinguish between leaf types. Neuffer and Bartel-heim, 1995 could not assign leaf types for 20 ± 5% of their samples.

Also in this thesis, assigning leaf types was difficult, because the fifteen individ-uals per biotype used for assigning leaf types varied in their appearance. Therefore,the most dominant leaf type was chosen to represent the whole biotype. This leadsto underestimation of rare leaf types, like heteris (Iannetta et al., 2007). This leaftype was assigned to some individuals of ID1117, indicating that the seed sample ofa single biotype consist of seeds with multiple genetic backgrounds.

When comparing the present leaf types with the phenological and morphological datapresented in section 4.1, some correlations between leaf type and functional traitscould be identified. For instance was the leaf type simplex associated with earlyflowering (figure 4.2(e)). This is in accordance with observations from Iannetta,2011 and Iannetta et al., 2007. However, simultaneously to assigning the attributesof early flowering, small plants and intermediate reproductive output to leaf typesimplex, cluster analysis also placed leaf type rhomboidea to this group (Iannettaet al., 2007). This is contradictory to the characterisation results in section 4.1. Here,ID140, assigned leaf type rhomboidea, was the complete opposite to ID1118 andID1089, assigned leaf type simplex.

Therefore, it can be concluded that leaf type still is an important trait to describeheterogeneity in a CAPBP population. Nevertheless, assigning it without geneticmarkers is a subjective process and association of leaf type to functional traits leadsto inconsistent results.

GerminationAs described in section 4.1.2, total germination of the resistant biotypes ID1120 andID1123 was poor at high and medium temperatures, compared to low temperatures.This was more pronounced for ID1120, compared to ID1123 (figure 4.7, table 4.2).Figures 4.8 and A.7 confirm the observation that ID1120 had low germination at25∘C. A possible explanation could be fitness costs due to resistance, since level ofresistance resembled the observed relationship: ID1120 had a higher RI than ID1123(table 4.6), therefore greater fitness costs and lower germination at 25 ∘C. Otherstudies on ALS resistant weeds found a generally lower germination rate for resistant,compared to susceptible biotypes at moderate and high temperatures (Dyer et al.,1993; Lamego et al., 2011; Schaedler et al., 2013). Further on, two studies onbroadleaf weeds (Kochia scoparia L. and Bidens subalternans) observed earlier andfaster germination of resistant, compared to susceptible biotypes at low temperatures(Dyer et al., 1993; Lamego et al., 2011). But the present results only showed acontinuously slower germination rate of resistant, compared to susceptible biotypes,independent on temperature table 4.2.

46 5 Discussion

The total percent germinated seeds of resistant and susceptible plants was similarin all three studies discussed (Dyer et al., 1993; Lamego et al., 2011; Schaedleret al., 2013). This is in contrast to the results presented in table 4.2, where theresistant biotypes had a significantly lower total germination at high and highertotal germination at low temperatures, compared to the susceptible biotypes. Theexplanation for the difference in germination rates, observed in the three studiescould be reduced feedback inhibition of ALS in resistant plants (Dyer et al., 1993).This lead to the presence of more long chain amino acids which were responsible forthe faster germination of resistant plants (Dyer et al., 1993). Now it is questionablewhether this explanation can also be applied to the phenomenon of enhanced totalgermination at low temperatures, observed here. Due to a lack of difference ingermination rate, it is probable that a different mechanism is responsible for thepresent observations.

In both germination experiments, ID1120, ID1123 and ID140 showed two flushes ofgermination. An external trigger causing the second flush can not totally be ruledout, as the seeds were taken out of the incubator whenever germination events wererecorded. Possible factors are therefore light and altered temperatures. However, allseeds of one experiment were always treated in the same way, including the timeoutside the incubator. Therefore, it can be argued that it is an internal trigger,specific for the biotype, that splits germination temporally.

In figure 4.8, it is striking that the three parameter log- logistic function is inappro-priate for correct modelling of germination curves of biotypes with two flushes ofgermination. This is because the function can only describe one slope. In order tocalculate a correct time point of 50% germination for this kind of data, a differentfunction is required. Consequently, the interpretation of a possible resistance costdiscussed earlier in this section might change. Germination in two flushes might befavourable in agricultural environments, where later germinating seeds might surviveweeding practices of the farmers.

Dose- response experimentThe dose- response curves were analysed with a three parameter log- logistic model.To prevent overfitting of the data, it was tried to reduce it to a two parameter log-logistic model, but due to the different upper limits of the biotypes, this was notpossible. Further, RIs with ED90 values were calculated (not shown). They did notchange the interpretation of the RIs calculated from ED50 values and were thereforenot included in this thesis.

Biotype ID1120 is the first case where a point mutation at position 574 of the ALSgene, leading to a substitution of tryptophane with serine, is described in a weedspecies (Tranel, Wright, and Heap, 2014; Yu et al., 2014). The common crossresistance pattern of point mutations at Trp574 is broad cross resistance to SU, TP

47

and IMI (Tranel and Wright, 2002; Yu et al., 2014). Research on Arabidopsisthaliana with an artificial mutation of Trp574-Ser confirms this pattern (Chang et al.,1998). In contrast to this, the present mutation in CAPBP only conferred resistanceto SU and TP (table 5.1). This confirms that cross resistance is not only dependenton the specific mutation but also on weed species (Yu et al., 2014). Though, it israther unexpected due to the close relation of CAPBP with A. thaliana. Nevertheless,it has to be kept in mind that CAPBP is a tetraploid weed species. Therefore, bothlevel and spectrum of resistance may differ from close related diploid species (Yuet al., 2014).

Table 5.1: Cross resistance pattern of ALS herbicides. R: resistant; S: susceptible

product active ingredient ID1120 ID1123

Primus florasulam (TP) R SLexus flupyrsulfuron (SU) R RHussar iodosulfuron (SU) R RPursuit imazethapyr (IMI) S S

In contrast to ID1120, the resistance mechanism causing resistance in ID1123 isnot yet identified. Considering target site mutations, Pro197 would be a suitablefit due to the lack of cross resistance to other ALS inhibitor chemical groups thanSU (Tranel and Wright, 2002; Tranel, Wright, and Heap, 2014). But thismutation was not found in both, the sequencing test of the parent plant (section 3.1)and the test of progeny (section 4.4). Sequencing results of section 4.4 excluded alsopoint mutations at position of Ala205, Asp376 and Arg377. On the basis of theseresults, there are only three earlier reported positions ALS TSR left: Ala122, Ser653and Gly654 (Tranel, Wright, and Heap, 2014).

Beside TSR, there is also the possibility that we are dealing with NTSR, and the mostprobably enhanced metabolism (section 2.3), causing resistance in ID1123. Metabolicresistance can be specific to a certain herbicide and is often characterised by a ratherlow level of resistance (Yu et al., 2014), matching the present observations. However,NTSR is only rarely reported in broad leaf weeds (Delye et al., 2011). But this mightbe due to a generally low number of studies on NTSR, compared to TSR (Yu et al.,2014). Metabolic resistance is mainly characterised by increased enzyme activity,which may cause allocation of resources from vegetative growth and reproduction todetoxification processes. In accordance with this, two studies conducted on fitnesscosts due to metabolic resistance to ALS herbicides, observed reduced vegetativegrowth of Lolium rigidium (Vila-Aiub et al., 2009[b]). In contrast to this, there wasno resistance cost in terms of vegetative growth found in the characterisation andcompetition experiments, discussed in section 5. The fact that there was no evidencefor fitness costs in the vegetative growth of CAPBP argues against the presenceof metabolic resistance in ID1123. However, a presence of metabolic resistance inID1123 can not be ruled out because of the entire lack of reports on fitness costs on

48 5 Discussion

broad leaf weed species.

As stated in section 4.2, analysis of the dose- response experiment gave evidence fordiffering cross resistance patterns of ID1120 and ID1123 (table 5.1). Together withthe lower resistance level of ID1123, compared to ID1120, this indicates that ID1123has a different resistance mechanism than the one identified in ID1120. However, thenature of this resistance mechanism is unknown and it can not be excluded that it isalso present in ID1120.

Competition experimentThe fact that it was possible to fit the same type of curve to the data of the compe-tition experiment as the dose- response data, shows how efficient crop density canbe used as an alternative weed defence strategy. The applied model demonstrates,that at a recommended sowing density of 250 plants per m2 (plantevaern-online,2014) weed biomass production was reduced by approximately 80% (table 4.9 andfigure 4.14). Due to a very high standard error, this result is not very reliable. But itcertainly gives an impression about the possibilities of competition as a strategy forintegrated weed management. This was recently confirmed by Olsen et al., 2012.Their field experiments showed, that increased crop density results in reduced biomassand increased crop biomass over several years (Olsen et al., 2012).

Plant fitnessAnalysis of resistant biotypes for fitness cost is mostly done to identify the natureof a fitness cost present under non-selective conditions and develop resistance man-agement tactics on this basis (Neve et al., 2009). These management tactics aimto shift the fitness advantage from the resistant back to susceptible biotypes. Con-sequently, susceptible biotypes would out-compete the resistant biotype and overa long period of time the proportion of resistant biotypes in the population would drop.

Vila-Aiub et al., 2009(b) reviewed the methodological and experimental require-ments to unequivocally measure fitness and fitness costs in plant populations. Manypublished studies cannot be interpreted because of the inappropriate experimental setup used (Neve et al., 2009; Vila-Aiub et al., 2009[b]). Therefore, both the qualityof the methodological and experimental prerequisites and the corresponding resultsare discussed in the following section.

First of all it is important to be able to compare the resistant and susceptiblebiotypes by having control of genetic background. The optimum would be isogeniclines (Neve et al., 2009). This was definitely not achieved here, but it can be assumedthat the genotype composition of CAPBP within a given family is constant (Neufferand Hurka, 1986). This is due to the fact that the rate of outcrossing in the speciesCAPBP is below 20% (Hurka and Neuffer, 1997; Shull, 1929). For ID140,ID1089 and ID970 the geographic origins are known and seeds were sampled within

49

a fairly small distance, resulting in genetically homogeneous composition of eachsample. ID1120 and ID1123 are each offspring of a single plant. In contrast to this,the exact origin of ID1117 and ID1118 is not known. The heterogeneous appearanceof leaf types of ID1117 was already discussed earlier in this section. Further, theboxplots on morphological traits of ID1118 showed large interquartile ranges, arguingfor heterogeneity also in this biotype, see figures 4.3 and 4.4. Nevertheless, thesebiotypes add valuable information to the assessment whether ID1120 and ID1123show decreased fitness. As stated in the beginning of this chapter, they make itpossible to account for general diversity of the species CAPBP.

The biochemical basis of the resistance mechanism is not entirely evident. Asdescribed in section 3.1, a heterogeneous point mutation on position Trp574 of theALS gene was identified in the parent plant of ID1120. But yet there is no informationabout the frequency of this mutation in the offspring present. In return, there wasevidence for cross resistance of ID1120 to SU and TP herbicides, whereas ID1123was only resistant to SU herbicides (section 4.2). As discussed earlier in this section,there is a possibility for multiple resistance mechanisms present that may influencefitness characteristics.

The third important point, that has to be taken into consideration when com-paring fitness traits of resistant and susceptible biotypes, is to analyse a number oflife- history traits. This was done in detail in the characterisation and germinationexperiment (section 4.1 and 4.1.2). The characterisation experiment did not showsignificant differences between susceptible and resistant biotypes. On the other hand,both germination experiments revealed a possible fitness cost due to resistance ofID1123 and ID1120.

Further, it is necessary to take competition for resources into consideration. Thiswas done here by analysing the competitive effect of SB on CAPBP plants. Thereby,competitive ability for space and light were measured. The experiment supplied noevidence on altered competitive ability of resistant, compared to susceptible biotypesat high nutrient supply.

Last but not least, Vila-Aiub et al., 2009(b) emphasises the importance of in-cluding investigation of the effect of environmental gradients on plant performance.The reason is that the fitness cost associated with resistance might be environmentallyspecific. The only gradient analysed was a temperature gradient at germination andit revealede a possible fitness cost of germination ability of resistant biotypes at hightemperatures.

In summary, the conducted experiments covered most of the criteria of a thoroughfitness assessment. The biggest uncertainty still is the nature of the biochemicalbasis of the resistance mechanism. Further, just a few environmental gradients havebeen assessed. Therefore, there might be other conditional fitness costs, next to poor

50 5 Discussion

germination at high temperatures. On the basis of this particular fitness cost it isdifficult to elaborate applicable resistance management practices, as temperature isa factor that is hard to manipulate. Moreover, the excellent germination of bothresistant biotypes at low temperatures might even be beneficial in practice. Temper-atures at sowing time in spring and autumn in Scandinavia often are in the rangeof alternating 10/17 ∘C, giving the resistant a fitness benefit over the susceptiblebiotype. In this context, it can only be speculated on the warming effect of climatechange, which could probably shift the fitness benefit back to the susceptible biotype.

Genetic analysisThe explanation for the small and overlapping peaks in the sequencing results ofprimer pair two could be the tetraploid nature of CAPBP. However, it seems thatgenetic variability between alleles of the biotypes analysed is limited in the rangeof primer pair one. Therefore, there are interpretable sequencing results for therange of amino acid position 190 to amino acid position 380 of the ALS genomeof ID1123 and ID1117. This is valuable information, as mutations at Pro197 arethe most common ALS point mutations recorded (Tranel, Wright, and Heap,2014). Nevertheless, the analysis was only a test and therefore just performed onone plant without replicates. In order to obtain reliable results, several plants ofthe two resistant biotypes should be analysed and replicated. Ideally, also the rangeof all eight point mutations, identified in CAPBP so far (Tranel, Wright, andHeap, 2014), should be covered. In this way it would be possible to adduce ev-idence on the whole spectrum of possible target site resistances of ID1120 and ID1123.

A solution to take care of the genetic variability between alleles of the tetraploidCAPBP could be to clone PCR products in vectors and amplify them in bacteria(Cui et al., 2012). In this way it is possible to separate the alleles and to examineonly one at a time when interpreting the sequencing results. In addition to this,PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR amplificationof specific alleles (PASA) are methods for point mutation analysis operating withoutsequencing (Corbett et al., 2008). In PCR-RFLP restriction endonucleases cut atspecific short nucleotide sequences, leading to fragments of different length dependenton presence or absence of the mutation in question. PASA is a modified PCR that isdependant on a primer that matches the resistance mutation. Under stringent PCRconditions, this primer only produces amplicons if the resistance is present (reviewedin (Corbett et al., 2008)).

CHAPTER 6Conclusion

In accordance with the first hypothesis, the resistant biotypes do not differ signifi-cantly from the susceptible biotypes. This is mainly due to the fact that there is abig diversity among susceptible biotypes and ID140 resembles ID1120 and ID1123 innearly all traits. Indeed, resistant biotypes were assigned different leaf types thansusceptible biotypes, but assigning leaf types is subjective and could not be linked tofunctional traits.

Cross resistance patterns of the Trp574-Ser mutation of ID1120 differ from thosereported so far for other weed species (Chang et al., 1998; Tranel, Wright, andHeap, 2014). ID1120 exhibits only cross resistance to TP and SU, whereas all othercases of mutations at Trp574 confer broad cross resistance, including IMI (Tranel,Wright, and Heap, 2014). Further, ID1123 shows no cross resistance to other ALSinhibiting herbicides than SU. Therefore, it can be concluded that ID1123 must havea different resistance mechanism than the one identified in ID1120. However, thenature of this resistance mechanism is unknown and it can not be excluded that it isalso present in ID1120. Overall, no multiple resistances to other herbicide classes arepresent in either ID1120 or ID1123. Therefore, the second hypothesis can be accepted.

The competition experiment did not provide evidence for reduced fitness of theresistant biotypes but illustrated the trade off between crop density and weed biomassproduction. Many other parameters influencing fitness have been assessed, main-taining most of the criteria for a proper assessment of fitness costs proposed byVila-Aiub et al., 2009(b). But there is still limited knowledge about the biochemicaland genetic background of the resistance of ID1120 and ID1123. Nevertheless, thediscussion on fitness costs due to resistance resulted in the identification of alteredtotal germination of both, ID1120 and ID1123. At high temperatures there is evidencefor a fitness cost and at low temperatures a fitness advantage of the resistant biotypes.This is in contrast to the third hypothesis, "There are no observable fitness differencesbetween resistant and susceptible biotypes", which therefore has to be rejected.

These results provide a clear picture of the effects of the recently found point mutationTrp574-Ser on the phenotype of CAPBP.

51

CHAPTER 7Perspectives

The field where the resistant CAPBP was found had a noninversion tillage systemwith cereals in four out of five preceding years. In order to insure good control ofresistant CAPBP integrated weed management should be adapted. In this particularfield this means that CAPBP has to be controlled with a different mode of actionthan ALS herbicides. To prevent further resistances from developing at least twomodes of action should be applied at the same time. Especially in winter wheatrotation of herbicides is difficult, as there are only two modes of action recommendedfor control of CAPBP at 3-4 leaf stage both in spring and autumn (Danish decisionsupport service, plantevaern-online, 2014). To enlarge the choice of herbicidemode of action, it would be necessary to adapt a more diverse crop rotation. As themechanism of resistance was not identified for ID1123 and the possibility of NTSR inID1120 could not be excluded, it might be possible that NTSR is present in the field.If this is the case, further use of herbicides for weed control may lead to continuousselection for resistant biotypes. In this way resistance level may increase and there isthe possibility of multiple resistance to develop (Yu et al., 2014). If it comes to this,the farmer may be solely reliable on non chemical weed control, such as ploughingand mechanical weeding. Consequently, a noninversion tillage system would no longerbe possible to maintain (Melander et al., 2013).

This thesis is another study out of many done on the consequences of resistancedevelopment in weed species. It is important to characterise new cases of resistancein order to understand the mechanisms of resistance. But this will not help toprohibit the further development of new resistances. The fact that the new pointmutation Trp574-Ser was found and that it exhibits different cross resistance patternsthan Trp574 mutations identified in other species shows, that on top of the onesalready reported, there are still possibilities for new, unpredictable resistances todevelop. Further on the amount of new compounds that are released to the market isdecreasing (Kudsk and Streibig, 2003) and there was only one new mode of actionfor weed control in cereals found during the last 20 years (Reddy et al., 2013). Thisreveals the need for sustainable use of the herbicides we have today. Here it is onthe farmers to embrace the principles of integrated weed management and follow the

53

54 7 Perspectives

guidelines set up to delay herbicide resistance development, available at the web siteof the herbicide resistance action committee (HRAC, 2014). Further, researchersshould focus on understanding the evolutionary factors behind resistance development(Neve et al., 2009). Only in this way it will be possible to minimise the developmentof new resistances.

Furthermore, there is a lot of practical work that could follow up on this thesis.First of all the characterisation of CAPBP plants germinated in autumn should befinished. There was no time to conduct a germination experiment with the seeds ofthese plants, but the results of this experiment may support or decline the findingsof the germination experiment conducted in spring.

The most important investigation tying up to this thesis is to analyse the genetic andbiochemical basis of resistance of ID1120 and ID1123. Hereby, both TSR and NTSRhave to be taken into consideration. Analysis for TSR could be done by repeating aPCR with the appropriate methods suitable for point mutation analysis of tetraploidspecies, described in section 5. NTSR could be investigated by measuring cytochromeP450 activity, as in the majority of cases of enhanced metabolism the activity of thisenzyme is enhanced (Yu et al., 2014).

Further, an appropriate function for modelling germination in two flushes should befound. With this function speed of germination could be calculated correctly. Thismight result in a revalidation of the altered fitness of the resistant, compared tothe susceptible biotypes analysed in this thesis. To get a broader picture on otherfitness costs possibly present in resistant biotypes of CAPBP, more environmentalgradients (eg. drought stress) should be analysed. With this increase in variables itwould be possible to apply multivariate statistics such as cluster analysis or principalcomponent analysis. This could increase the possibility to differentiate betweenbiotypes and identify variation in fitness. But if this is done, it would be essential tohomogenise the genetic background of the single biotypes. This could be done byconducting trials with progeny of plants that were protected from pollen of otherbiotypes.

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List of Figures

1.1 Resistance development of herbicides with different modes of action(Heap, 2014) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1 Four leaf types of the species CAPBP. . . . . . . . . . . . . . . . . . 52.2 Pathway of ALS biosynthesis. . . . . . . . . . . . . . . . . . . . . . . 72.3 Target site mutations conferring resistance to ALS inhibiting herbicides. 8

3.1 Set-up of the Competition experiment. . . . . . . . . . . . . . . . . . 183.2 Example of a curve of a four- parameter logistic model. . . . . . . . 22

4.1 Correlograms of experiment two. They show the correlation betweenspecific phenological and morphological traits of CAPBP. . . . . . . 24

4.2 Boxplots on the duration from sowing to specific growth stages. . . . 254.3 Boxplots on plant dry weight at different development stadia. . . . . 274.4 Boxplots on morphological traits. . . . . . . . . . . . . . . . . . . . . 284.5 Boxplots on seed characteristics. . . . . . . . . . . . . . . . . . . . . 284.6 Leaf types of six CAPBP biotypes. . . . . . . . . . . . . . . . . . . . 294.7 Overview of all germination curves of four CAPBP biotypes at three

different temperatures and different concentrations of Potassium Nitrate. 314.8 Germination curves of four susceptible and two resistant biotypes. . 324.9 Dose- response curves with relative fresh weight of all ALS inhibit-

ing herbicides used in this experiment: Primus (florasulam), Lexus(flupyrsulfuron), Hussar (iodosulfuron), Pursuit (imazethapyr). . . . 34

4.10 Dose- response curves with relative fresh weight of Starane (fluroxypyr),Glyfonova (glyphosate) and Fighter (bentazon). It is not possible tofit a curve to the data of Stomp (pendimethalin). . . . . . . . . . . . 36

4.11 Harvest of the competition experiment. . . . . . . . . . . . . . . . . 384.12 Dry weight of the target CAPBP plants at different densities of spring

barley of the first and the second competition experiment. . . . . . . 384.13 Dry weight of four different CAPBP plant lines at different densities

of spring barley (SB). . . . . . . . . . . . . . . . . . . . . . . . . . . 394.14 Model on CAPBP dry weight at different densities of spring barley (SB). 404.15 Results of the PCR with primer pair one. . . . . . . . . . . . . . . . 414.16 Alignment of the sequencing results of Primer pair one. . . . . . . . 41

61

62 List of Figures

A.1 Boxplots on the duration from sowing to specific growth stages ofcharacterisation experiment one. . . . . . . . . . . . . . . . . . . . . 66

A.2 Boxplots on plant dry weight at different development stadia of char-acterisation experiment one. . . . . . . . . . . . . . . . . . . . . . . . 67

A.3 Boxplots on plant fresh weight at different development stadia ofcharacterisation experiment one. . . . . . . . . . . . . . . . . . . . . 68

A.4 Boxplots on plant fresh weight at different development stadia ofcharacterisation experiment two and three. . . . . . . . . . . . . . . 69

A.5 Boxplots on morphological traits of characterisation experiment twoand three. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

A.6 Mature CAPBP plants just before harvest. . . . . . . . . . . . . . . 71A.7 Germination curves of the first characterisation experiment. . . . . . 73A.8 Pictures of the spraying results with ALS inhibitors. . . . . . . . . . 74A.9 Pictures of the spraying results of herbicides with other modes of action

than ALS inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

List of Tables

3.1 Seeds used for the experiments. . . . . . . . . . . . . . . . . . . . . . 113.2 Germination rate of the first experiment and experimental setup of all

characterisation experiments. . . . . . . . . . . . . . . . . . . . . . . . 143.3 Herbicides and doses for the susceptible and resistant biotypes. . . . 173.4 Primers of the amplification of two ALS gene fragments. . . . . . . . 193.5 Master mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4.1 Number of days to 50% germination of five CAPBP biotypes at threetemperatures and a concentration of 2 g/l KNO3. . . . . . . . . . . . 30

4.2 Comparison of resistant and susceptible biotypes of the germinationconditions experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . 30

4.3 Number of days to 50% germination and the total percentage germi-nated of five CAPBP biotypes. . . . . . . . . . . . . . . . . . . . . . . 33

4.4 Comparison of the number of days to 50% germination between sus-ceptible and resistant biotypes. . . . . . . . . . . . . . . . . . . . . . 33

4.5 ED50 of all ALS inhibiting herbicides investigated. . . . . . . . . . . 344.6 Resistance indices of treatment with ALS inhibiting herbicides. . . . 354.7 ED50 values of treatment with Starane, Glyfonova and Fighter. . . . 354.8 Resistance indices of Starane, Glyfonova and Fighter. . . . . . . . . . 364.9 Spring barley densities with 50, 80 and 90 percent of weed biomass

reduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.1 Cross resistance pattern of ALS herbicides. . . . . . . . . . . . . . . . 47

A.1 Number of days to 50% germination and the total percentage germi-nated of five CAPBP biotypes of germination experiment one. . . . . 72

63

APPENDIX AFirst chapter of appendix

A.1 CharacterisationA.1.1 Characterisation experiment oneFigure A.1 shows the phenological traits investigated in characterisation experimentone. In the early growth stages up to four leaf stadium, ID140 developed fastest. Af-terwards, ID140 was not longer significantly different from the two resistant biotypes,ID1120 and ID1123. This resembles the results of characterisation experiment twoand three, presented in the main part of the thesis.

Morphological traitsFigures A.2 and A.3 show the morphological traits investigated in characterisationexperiment one. Due to bad germination, there were not enough plants to measureevery trait at all the plants. Therefore there are missing measurements in both figures.For both fresh and dry weight measurements, there was significant difference betweenthe susceptible ID1117 and the resistant biotypes ID1120 and ID1123. However,whenever there was a measurement of ID140 present, it was not significantly differentfrom the resistant biotypes.

65

66 A First chapter of appendix

ID140 ID1117 ID1089 ID1120 ID1123

1015

20

Day

s to

Ger

min

atio

nb

a

a

a

a

(a) Days to germination.

ID140 ID1117 ID1089 ID1120 ID1123

1015

2025

Day

s to

2 le

af s

tadi

um

b

a

a

a

a

(b) Days to two leaf stadium.

ID140 ID1117 ID1089 ID1120 ID1123

2025

30

Day

s to

4 le

af s

tadi

um b

a

a

ab

a

(c) Days to four leaf stadium.

ID140 ID1117 ID1089 ID1120 ID1123

5060

7080

Day

s to

flow

erin

g pr

imor

dium

ab

a

b b

(d) Days to flowering primordium.

ID140 ID1117 ID1089 ID1120 ID1123

4050

6070

80

Day

s to

sta

rt o

f flo

wer

ing

bc

b

a

c

c

(e) Days to flowering.

Figure A.1: Boxplots on the duration from sowing to specific growth stages of char-acterisation experiment one. Biotypes marked with different letters are significantlydifferent from each other.

A.1.2 Characterisation experiment two and threeFigure A.4 shows fresh weight measurements of characterisation experiment two andthree. The interpretation is similar to the dry weight measurements, presented insection 4.1. Figure A.5 shows rosette diameter, LA and LN at rosette stadium andat the end of the growth period.

A.1 Characterisation 67

ID140 ID1117 ID1120 ID1123

01

23

4

Dry

wei

ght a

t ros

ette

sta

dium

(g)

aab

b

(a) Dry weight at rosette stadium.

ID140 ID1117 ID1089 ID1120 ID1123

02

46

810

Dry

wei

ght a

t flo

wer

ing

(g)

b

a

b

b

(b) Dry weight at flowering.

ID140 ID1117 ID1089 ID1120 ID1123

05

1015

2025

Dry

wei

ght a

t see

d pr

oduc

tion

(g)

a

bb

(c) Dry weight of the leaves at the end ofthe growth phase.

ID140 ID1117 ID1089 ID1120 ID1123

010

2030

4050

60

Dry

wei

ght o

f the

flow

er (

g)

b

a

a

(d) Dry weight of the flower at the end ofthe growth phase.

Figure A.2: Boxplots on plant dry weight at different development stadia of character-isation experiment one. Biotypes marked with different letters are significantly differentfrom each other.


ID140 ID1117 ID1120 ID1123

010

2030

40

Fre

shw

eigh

t at r

oset

te s

tadi

um (

g)

a a

a

(a) Fresh weight at rosette stadium.

ID140 ID1117 ID1089 ID1120 ID1123

020

4060

80

Fre

shw

eigh

t at f

low

erin

g (g

)

b

a

b b

(b) Fresh weight at flowering.

ID140 ID1117 ID1089 ID1120 ID1123

050

100

150

Fre

shw

eigh

t at s

eed

prod

uctio

n (g

)

a

b

b

(c) Fresh weight at the end of the growthphase.

ID140 ID1117 ID1089 ID1120 ID1123

050

100

150

200

Fre

shw

eigh

t of t

he fl

ower

(g)

b

ab

a

(d) Fresh weight of the inflorescence atthe end of the growth phase.

Figure A.3: Boxplots on plant fresh weight at different development stadia of char-acterisation experiment one. Biotypes marked with different letters are significantlydifferent from each other.


010

2030

40

Fre

sh w

eigh

t at r

oset

te s

tadi

um (

g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

dee

ab

aa a

cd

bc

de

e

(a) Fresh weight at rosette stadium.

020

4060

8010

012

0

Fre

sh w

eigh

t at f

low

erin

g (g

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

ce

e

bd

be

bc

bc

ab

cdecde

cde

ce

(b) Fresh weight at flowering.

050

100

150

Fre

sh w

eigh

t at t

he e

nd o

f the

gro

wth

pha

se (

g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

de

be

abc

bd

ade

ab ab a

cde

e

de

e

(c) Fresh weight at the end of the growthphase.

050

100

150

Fre

sh w

eigh

t of t

he in

flore

scen

ce (

g)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

c

c c

bc

ac ac

ab

a

bc

a ab ab

(d) Fresh weight of the inflorescence at theend of the growth phase.

Figure A.4: Boxplots on plant fresh weight at different development stadia of charac-terisation experiment two and three. For each biotype, the results of the spring sown(blue) and autumn sown (white) experiments are shown separately.


2040

6080

Ros

ette

dia

met

er a

t ros

ette

sta

dium

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

bcbc

ab

a

a a

bc bc

bc

c

(a) Rosette diameter at rosette stadium.

1020

3040

5060

Ros

ette

dia

met

er a

t the

end

of t

he g

row

th p

hase

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

bd bd

ab

bc

ab

aa

dcd

cdcd

(b) Rosette diameter at the end of thegrowth period.

020

040

060

080

010

00

Leaf

are

a at

ros

ette

sta

dium

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

cdd

b

aa a

cd

c

cd

c

(c) Leaf area at rosette stadium.

050

010

0020

00

Leaf

are

a at

the

end

of th

e gr

owth

pha

se (

cm)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

cd

bd

ab

bc

aa a

d

cd

d

cd

(d) Leaf area at the end of the growth pe-riod.

1020

3040

5060

70

Leaf

num

ber

at r

oset

te s

tadi

um (

cm)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

c

c

b

aa a

c

bc

c

bc

(e) Leaf number at rosette stadium.

010

020

030

040

0

Leaf

num

ber

at th

e en

d of

the

grow

th p

hase

(cm

)

ID140 ID1117 ID1118 ID1089 ID1120 ID1123

cdcd

ab

bc

aa a

cd

d

cd

d

(f) Leaf number at the end of the growthperiod.

Figure A.5: Boxplots on morphological traits of characterisation experiment two andthree. For each biotype, the results of the spring sown (blue) and autumn sown (white)experiment are shown separately. Biotypes marked with different letters are significantlydifferent from each other.


Figure A.6: Mature CAPBP plants just before harvest. It is presented from left toright: ID140, ID1117, ID1118, ID1089, ID1120, ID1123.


A.1.3 Germination experiment oneFigure A.7 and table A.1 show the results of germination experiment one. ID140 hasthe highest germination rate, followed by the two resistant biotypes. ID1089 andID1117 have a very low total germination of below 20 %.

Table A.1: Number of days to 50% germination and the total percentage germinatedof five CAPBP biotypes of germination experiment one. The seeds were germinated at25∘C and a concentration of 0.1% KNO3.

Days until 50% germination Total percentage germinated

ID1089 1.36 ± 0.054 18.68 ± 0.013ID1117 3.22 ± 0.149 13.95 ± 0.009ID1120 4.37 ± 0.180 33.95 ± 0.015ID1123 2.33 ± 0.073 39.93 ± 0.013ID140 23.17± 100±

A.2 Dose-response 73

1 10

0.0

0.1

0.2

0.3

0.4

0.5

0.6ID1089ID1117ID1120ID1123ID140


Per

cent

ger

min

ated

Figure A.7: Germination curves of the first characterisation experiment. The graphshows three susceptible (ID140, ID1117, ID1089) and two resistant (ID1120, ID1123)CAPBP biotypes.

A.2 Dose-responseFigure A.9 shows pictures of representable plants of each herbicide treatment 28 daysafter treatment.


(a) Primus (b) Lexus

(c) HussarOD (d) Pursuit

Figure A.8: Pictures of the spraying results with ALS inhibitors.

(a) Starane (b) Glyfonova

(c) Fighter (d) Stomp

Figure A.9: Pictures of the spraying results of herbicides with other modes of actionthan ALS inhibition.

characterisation of a capsella bursa- pastoris biotype resistant …library.au.dk/fileadmin/ ·...

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