Mechanisms of herbicide resistance 1221 Target site resistance

As detailed elsewhere, herbicides have distinct target sites where they act to disrupt biochemical processes leading to cell, tissue and plant death. The majority of target sites are enzymes and the interaction between herbicide and target site can be disrupted if there is a change in the primary structure of the enzyme protein molecule. Where this occurs, the herbicide may no longer be effective in blocking the action of the target site and the plant will not die, but exhibit herbicide resistance (Figure 12.1).

Target site resistance is often the result of a single point mutation in the gene coding for the target site, so is the result of a change in one nucleic acid in the

Figure 12.1 Diagrammatic representation of Target Site Resistance. TS, target site; H, herbicide.

target site gene, resulting in one amino acid change in the final target site protein. Examples are given below. Target site-mediated resistance to ACCase-inhibiting herbicides

A number of mutations in the acetyl-CoA carboxylase (ACCase) gene have been reported in grasses, which result in target site resistance to ACCase-inhibiting herbicides. Different cross-resistance patterns are noted for each mutation (Devine, 1997; Devine and Shukla, 2000; Delye, 2005). An isoleucine-to-leucine substitution within the carboxytransferase region of ACCase confers resistance in Avena fatua (Christoffers et al., 2002), Lolium rigidum Gaud. (Zagnitko et al., 2001; Delye et al., 2002a; Tal and Rubin, 2004), Setaria viridis (L.) Beauv. (Delye et al., 2002b) and Alopecurus myosuroides Huds (Delye et al., 2002a). In A. myosuroides this mutation is designated I1781L (Delye et al., 2002a) indicating the position where the amino acid substitution has taken place. In all cases this mutation confers cross-resistance to all fops and most dims. Another mutation (Ile 2041 ^ Asn), also in the carboxytransferase region of ACCase, in L. rigidum and A. myosuroides has been reported, which confers resistance to fops but not to dims (Delye et al., 2003). These observations may increase our understanding of the differences in herbicide binding to ACCase between fops and dims. Target site-mediated resistance to ALS-inhibiting herbicides

Many of the cases of herbicide resistance to acetolactate synthase (ALS) inhibitors reported to date are caused by an altered target site rather than due to enhanced metabolism. Five naturally occurring mutations are found in native populations that give rise to resistance. Many others are found in bacteria and yeast systems. Mutations are found in two regions: A; amino acid sequences 124-205 and B; amino acid sequences 574-653 (Gressel, 2002; Table 12.1). This is not the catalytic site of the enzyme but a separate herbicide-binding site. Different patterns of cross-resistance are noted for each of these amino acid substitutions, and the flexibility of substitution while still maintaining enzymic activity has probably contributed to the rapid rise in resistance to this class of herbicide. Resistance to imidazolinones, conferred by Ser670 ^ Asp or Ala122 ^ Thr, does not confer very high levels of resistance to the other classes of ALS inhibitor (Sathasivan et al., 1990, 1991; Bernasconi et al., 1995). Studies with ALS-resistant Arabidopsis thaliana mutants csr1-1 (Pro197 ^ Ser) and csr1-2 (Ser653 ^ Asn) identified complex cross-resistance patterns with respect to 22 ALS-inhibiting herbicides (Roux et al., 2005) . This suggests complex and non-ubiquitous binding characteristics for this herbicide family. Target site-mediated resistance to Photosystem II-inhibiting herbicides

In 1970 Ryan reported the failure to control groundsel (Senecio vulgaris) with simazine and atrazine in a nursery in Washington State, USA. These residual herbicides had been used once or twice a year in the nursery from 1958 to 1968. Susceptible plants grown from seeds collected from a location where triazines had not been in continuous use were adequately controlled by 0.56-1.12kgha-1, but seedlings from the nursery were barely

Table 12.1 A selection of mutations that confer target site resistance to ALS (modified from Gressel, 2002). In some cases different cross-resistance patterns have been reported for a single mutation by different researchers.


Gene name

Selector and cross-resistances

Little or no cross-resistance

Met124 ^ Glu


Met124 ^ Ile


Ala155 ^ Thr



Pro197 ^ His

SU, some IM

Pro197 ^ Gln


SU, some IM

Pro197 ^ Ala


SU, some IM

Pro197 ^ Ser




Prc>197 ^ Arg

SU, some IM

Pro197 ^ Leu

SU, some IM

Pro197 ^ Thr

SU, some IM

Arg199 ^ Ala

Arg199 ^ Glu

Ala205 ^ Asp

Gln269 ^ His


Asn522 ^ Ser

Tro574 ^ Leu



Trp574 ^ Ser


Trp574 ^ Phe


Ser653 ^ Asn

Csr l-2, imr



Ser653 ^ Thr



Ser653 ^ Phe


SU, sulfonylurea; TP, triazolopyrimidine sulfonamide; IM, imidazolinone; PC, pyrimidyloxybenzoate.

SU, sulfonylurea; TP, triazolopyrimidine sulfonamide; IM, imidazolinone; PC, pyrimidyloxybenzoate.

affected, even by doses as high as 17.92 kg ha-1. Triazine resistance is now known to have evolved independently throughout the world due to persistent and prolonged use in monocultures in orchards, vineyards, nurseries and maize crops. Cross-resistance is commonly observed (Table 12.2).

A single mutation in the psbA gene coding for a 32-kD protein that forms part of the Photosystem II (PS II) complex in the thylakoids can lead to resistance to the herbicides that inhibit photosynthesis at PS II (Oettmeier, 1999). These single mutations cause resistance to a number of PS II inhibiting herbicides to varying degrees (see Table 5.2). In some cases 1000 times more herbicide is needed to displace QB from this site. As this binding site is common to many PS II inhibiting herbicides the cross-resistance observed is not surprising. However, cross-resistance does not always extend to all PS II herbicides as is clearly demonstrated in Table 5.2 . The mutation Ser264 ^ Gly results in decreased binding and efficacy of triazines but not other classes of PS II inhibitors. The mutation Ser264 ^ Thr results in a broader spectrum of resistance to phenylureas and the triazine herbicides, for example linuron and atrazine, respectively. Resistance to diuron and metribuzin is conferred by the mutation Val219 ^ Ile. In addition to these mutations of the D1 protein, Ala251 ^ Arg and Val280 ^ Leu are also found in weed populations in the wild. In addition laboratory studies have also identified a further mutation, Ser268 ^ Pro, that also confers resistance. It is interesting to note that there is a fitness price to pay for these mutations to the D1 protein, with reduced CO2 fixation, quantum

Table 12.2 Photoreduction of DCPIP by thylakoids isolated from atrazine-resistant and susceptible biotypes of oilseed rape (Brassica napus L. cv. Candelle) (A. Cobb, unpublished observations).


Resistant (R)

Susceptible (S)



7.5 x 10-4

1.0 x 10-8



1.0 x 10-5

2.0 x 10-7



1.75 x 10-6

6.5 x 10-8



9.0 x 10-6

1.8 x 10-6



8.0 x 10-5

6.0 x 10-5



2.6 x 10-7

3.5 x 10-7



1.1 x 10-7

0.8 x 10-6


Values are the molar concentration of herbicide needed to inhibit the photoreduction of DCPIP by 50% (I50). The resistance ratio I50R/I50S reflects the degree of resistance measured in the assay.

Values are the molar concentration of herbicide needed to inhibit the photoreduction of DCPIP by 50% (I50). The resistance ratio I50R/I50S reflects the degree of resistance measured in the assay.

yield and biomass accumulation, all reported for biotypes possessing a mutation to the D1 protein that results in herbicide resistance. The mechanism of this fitness price has not been satisfactorily elucidated to date, but it has been shown to affect electron flow, chlorophyll a: b ratios and to increase damage in high-intensity light conditions (Holt and Thill, 1994). Target site-mediated resistance to Photosystem l-diverting herbicides

There have been only limited reports of resistance to this class of herbicide and little is known regarding the resistance mechanisms responsible. No evidence of target-site resistance involvement has yet been found and it is thought that most cases of resistance are a result of enhanced herbicide metabolism or sequestration away from the site of action. In Lolium perenne and Conyza bonariensis increased levels and activities of enzymes that detoxify active oxygen species have been measured in resistant biotypes. These are superoxide dismutase, ascorbate reductase and glutathione reductase (Shaaltiel and Gressel, 1986). Other studies with C. bonariensis have suggested the immobilisation of paraquat in resistant biotypes, possibly by binding to cell wall components, so that less of the herbicide can reach the thylakoid. Alternatively, paraquat uptake and movement may be reduced in resistant biotypes of Hordeum glaucum. Target site-mediated resistance to cell division inhibitors

Dinitroanilines have been used annually for approaching three decades in the cotton fields of the USA and on oilseeds and small grain cereals in the Canadian prairies. It was therefore not unexpected when resistance was reported in 1984 for Eleusine indica (L.) Gaertn. (goosegrass), in 1989 for Setaria viridis (green foxtail) and in 1992 for Sorghum halepense (Johnsongrass), as detailed by Smeda and Vaughn (1994). In E. indica the resistant biotype has shown cross-resistance to all dinitroanilines, dithiopyr and amiprophos-methyl, but sensitivity to propyzamide and chlorthal-dimethyl, and enhanced sensitivity to the car-bamates (Vaughn et al. : 1987). Furthermore, there seems to be no differences in fitness between the resistant and susceptible biotypes.

In resistant biotypes of S. viridis and E. indica a specific base change in the sequence of the TUA1 gene causes an amino acid substitution in a-tubulin from threonine to iso-leucine at position 239. This results in a conformational change to the surface of the a-tubulin molecule, which prevents herbicide binding.

tn both species, resistance is controlled by nuclear, recessive genes that are mutant alleles of an a-tubulin gene that causes a substitution of either Thr239 to Ile, or Met268 to Thr in E. indica or Leu136 to Phe in S. viridis. Thr239 ^ Ile is reported to give rise to resistance to the dinitroanaline herbicides, whereas Met268 ^ Thr gives rise to low levels of trifluralin resistance (Vaughn et al., 1990; Yamamoto et al., 1998). Target site-mediated resistance to glyphosate

For such an extensively used herbicide, there have been surprisingly few reports of glyphosate-resistant weed populations. The mode of action of glyphosate is to inhibit the enzyme EPSP synthase. A single mutation in the gene encoding this enzyme is reported to have resulted in glyphosate resistance in Eleusine indica in Malaysia (Lee and Ngim, 2000). The same mutation (Pro106 ^ Thr) has also been reported in the EPSP synthase gene from herbicide-resistant Lolium rigidum in Australia (Wakelin and Preston, 2006). A further case of glyphosate resistance appears to be due to an increase in the transcription of EPSP synthase, resulting in twice the amount of enzyme (Gruys et al., 1999). There have been many reports of glyphosate-resistant bacterial EPSP synthase and these have proved useful in the development of glyphosate-tolerant crops (see Chapter 13). Target site-mediated resistance to auxin-type herbicides

Although auxin-t ype herbicides have been extensively used for over 60 years there are few reported examples of weeds that are resistant to them. This is not too surprising - as these herbicides are generally foliartapplied and nontpersistent, it is difficult to envisage the generation of the major selection pressures needed to favour the evolution of resistant weeds. However, in 1985 Lutman and Lovegrove demonstrated an approximate tentfold difference in sensitivity to mecoprop in two populations of chickweed (Stellaria media), the more resistant population being found in grassland where mecoprop was seldom used. In subsequent studies Lutman and colleagues suggested that resistance was not due to differences in uptake or movement of mecoprop, but that more of the herbicide was apparently immobile in resistant plants, presumably bound to structural polymers (Coupland et al., 1990). An alternative hypothesis has been proposed by Barnwell and Cobb (1989) and Cobb et al. (1990). Their studies confirmed the original observations of resistance by Lutman and Lovegrove (1985) and reported reduced vigour in resistant plants (Figure 12.2). Furthermore, an investigation of Ht-efflux induced by mecoprop in etiolated S. media stems revealed that 170,000 times more mecoprop was needed in resistant plants to induce an amount of efflux equivalent to that obtained from susceptible plants. Such differences in mecoprop binding could imply an alteration in the auxin receptor in resistant plants that may also contribute in mecoprop resistance in this important weed species.

Further examples of increased tolerance of weeds to auxin-type herbicides have been observed in New Zealand and reported by Popay and colleagues (1990) . These include

Mecoprop (kg active ingredient ha 1)

Figure 12.2 Mecoprop resistance in Stellaria media (chickweed). (a) Plants photographed from above. (b) ED50 graph. o, WRO (susceptible); •, BAO (resistant). (After Barnwell and Cobb,1989.)

Mecoprop (kg active ingredient ha 1)

Figure 12.2 Mecoprop resistance in Stellaria media (chickweed). (a) Plants photographed from above. (b) ED50 graph. o, WRO (susceptible); •, BAO (resistant). (After Barnwell and Cobb,1989.)

populations of nodding thistle (Caruns nutans) requiring 5 to 30 times more MCPA and 2,4-D than expected, and populations of giant buttercup (Ranunculus aris) being tolerant of 4.8 times the dose of MCPA compared with susceptible populations. Since uptake, translocation and enhanced metabolism have been eliminated as resistance mechanisms in some cases of resistance to auxin analogues, then it seems a distinct possibility that mutations in the auxin-binding protein might be involved in the resistance (Peniuk et al., 1992). However, mutation in the auxin-binding protein has only been confirmed in one case, Sinapis arvensis. In this case the auxin receptor did not bind auxin-type herbicides. However, it also did not bind endogenous auxins either and this represents a severe fitness price for possessing herbicide resistance. Plants containing the insensitive auxin receptor lacked apical dominance and, in the absence of auxin-type herbicides, were very uncom-petitive due to their heavily branched growth habit and their lack of vertical growth (Deshpande and Hall, 2000; Webb and Hall, 1999). Target site-mediated resistance to cellulose biosynthesis inhibitors

There have been no reports of resistance in the field to the cellulose biosynthesis inhibitors (CBIs). Equally, the generation of resistant mutants of Arabidopsis in the laboratory has been difficult (Vaughn, 2002). Selection from extensive mutant screens eventually generated isoxaben- and dichlobenil-resistant plants, which were not cross-resistant to either flupoxam or isoxaben, indicating that the three herbicides did not share a common site. Furthermore, none of the mutants exhibited enhanced herbicide metabolism. Vaughan (2002) reports that at least one of the isoxaben-resistant mutants has an alteration in a cellulose synthase gene. Further confirmation of this finding at the gene sequence is awaited.

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