Separation of the water soluble

Following chromatographic separation of the ftrst two samples of water soluble compounds, it was discovered that over 90% of the radioactivity had been lost during sample preparation. Losses of radioactivity were subsequently monitored closely throughout the preparation of the other four samples chromatographed. The monitoring confmned that most of the radioactivity was being lost during sample preparation for both resistant and susceptible plants (Table 9. 1 3). Further

investigations indicated that large losses were occurring whenever solutions were evaporated to dryness, with the radioactivity apparently disappearing into the rotary evaporator. Only some of this radioactivity could be recovered subsequently from vessels within the evaporator. A chemist at the OowElanco laboratory in New Plymouth contacted about this problem stated they avoid evaporating solutions of phenoxy chemicals to dryness as they have experienced similar losses of material (J. Cowles, pers comm). Time constraints prevented development of more satisfactory sample preparation techniques to permit further analysis of water soluble compounds.

Table 9.13: Losses of radioactivity recorded during the processing of samples containing water soluble compounds from two replicates of resistant and susceptible nodding thistle plants.

Resistant Susceptible

plants plants

Rep 2 Rep 3 Rep 2 Rep 3

Radioactivity in water

soluble fraction (Bq) 5029 4396 2896 2504 % lost during butanol step 23.3 38.9 33.7 41 .7 % remaining water soluble

following �-glucosidase 20.5 1 6.7 1 4. 1 23.7 % lost producing 90%

ethanol solution 46.9 8 1 .2 55.0 23.8

% lost producing acetone

solution 69.8 41 .3 66.7 31 .3 Radioactivity in acetone solution (Bq) 453 2 1 5 227 453 Total % loss of radioactivity 91 .0 95. 1 92.2 8 1 .9 Mean 3706 34.4 .1 8.7 51 .7 52.3 337 90.0

The large losses of radioactivity meant valid comparisons of compounds obtained from susceptible and resistant plants was not possible. The low quantities of radioactivity remaining after sample preparation also resulted in poor quality chromatograms. However some chromatograms did indicate that at least three different water soluble compounds containing radioactivity were present in the roots of the nodding thistle plants. The Rf value of a major compound was the same as for the 2,4-0 applied to chromatograms as a reference point, suggesting that some 2,4-0 was conjugated to sugar without fIrst being hydroxylated. Formation of such glucose esters of 2,4-0 commonly occurs in plants (Ashton and Crafts 1 98 1 ).

9.4.4 Conclusions

concentrations of unmetabolised 2,4-D molecules 7 days after application than resistant plants. The 2,4-D molecules were probably converted more rapidly in the resistant plants into both water soluble and ether soluble metabolites. Details of these breakdown pathways could not be ascertained due to difficulties with sample

preparation.

9.5 DISCUSSION

Pillmoor and Gaunt ( 198 1 ) have discussed the difficulty of assessing the exact mechanism of selectivity with phenoxy herbicides. Both amino acid conjugates and sugar esters of 2,4-D can easily convert back to active 2,4-D molecules within the plant. Resistant plants may differ in sensitivity from susceptible plants because the equilibrium between active and inactive fonns of 2,4-D differs, with resistance resulting from concentrations of active herbicide molecules being maintained below toxic levels in sensitive tissues.

Further research could be usefully conducted on 2,4-D metabolism in nodding thistle by monitoring the changes in metabolite levels throughout the days following

application, rather than just at 7 days after spraying. Likewise, studies of metabolism in tissues other than just the roots would assist with interpretation of results.

Many similarities exist between the resistance to phenoxy herbicides in nodding thistle and giant buttercup. For example, the magnitude of resistance is similar between the two species, and the selection pressure which resulted in the resistance was probably also similar (Bourdot et aI 1990). Since the experiments with radiolabelled 2,4-D were completed with nodding thistle, similar experiments have been conducted with 1 4C_ MCPA on the phenoxy-resistant giant buttercup (McNaughton 1 991). This study also showed no difference between susceptible and resistant biotypes in foliar penetration of herbicide. McNaughton concluded that resistance in giant buttercup resulted from a combination of reduced translocation to the stolons and decarboxylation of MCPA. The MCPA molecules used in the giant buttercup work contained 14C only in the carboxyl side-chain, unlike the ring-labelled molecules used in our work. Large quantities of radioactivity were lost from the giant buttercup tissue, and in some cases there was more lost from resistant than susceptible plants. Although no 1 4C could be detected in the air near treated tissue, it was assumed the side chain was being cleaved from the MCPA molecule and radioactivity was lost through subsequent production of

14C02' This fonn of phenoxy degradation has been detected in a num ber of plant species (Pillmoor and Gaunt 198 1).

If decarboxylation had occurred in our experiments, radioactivity would not have been lost. The 14C atoms would have remained in the resulting phenol metabolite, and this molecule in turn would have been conjugated with glucose to become water soluble (Pillmoor and Gaunt 198 1). Our results were consistent with this scenario because a higher quantity of water soluble materials were detected in resistant plants (Section 9.4.3.1). However further work would be required to prove it, either by repeating the experiments using chain-labelled 2,4-0, or improving the identification procedure for the water soluble compounds.

Whether increased rates of degradation involved decarboxylation, hydroxylation, amino acid conjugation or some other metabolic pathway, presumably there were greater quantities of the enzyme necessary to control this metabolism within the

resistant plants. Very little is known of the enzymes responsible for the metabolism of the phenoxyacetic acids, though experiments have suggested that inducible enzymes may be involved in some cases (Pillmoor and Gaunt 198 1 ). This increase in enzyme quantity over time as a result of exposure to herbicide would be consistent with observations in our trials. There was usually no difference between resistant and susceptible plants in severity of symptoms for the first few days after treatment, then the resistant plants recovered and the susceptible plants continued to die. An inducible enzyme system that has been implicated with detoxification of foreign molecules in plants is the microsomal cytochrome P450 monooxygenase system, which has been shown to catalyze hydroxylation of 2,4-0 (Jones and Casely 1989).

Hydroxylation of the phenyl ring followed by glucose conjugation has been shown recently to be a major metabolism pathway for tribenuron-methyl in wheat (Ryan and Oulka 1 990). If cross-resistance to this herbicide did exist in nodding thistle, as suggested by results reported in Chapter 5, this would be explained by the presence of an enzyme system which catalyses hydroxylation.

An understanding of resistance mechanisms for herbicides in plants can occasionally assist with designing control strategies for such plants. For example, if resistance was caused by poor penetration of cuticles, the addition of a surfactant to the herbicide could help overcome this resistance. One possible strategy for phenoxy-resistant nodding thistle could be to prevent the functioning of the enzyme system responsible for deactivating the herbicides. Research has shown that the activity of cytochrome P450 can be blocked by several chemicals (eg aminobenzotriazole), thus inhibiting the metabolism of some herbicides (Jones and Casely 1 989). If this enzyme system was responsible for herbicide resistance in nodding thistle, the addition of a chemical such as aminobenzotriazole to the herbicide applied to weeds could overcome the

resistance.

One chemical readily available in New Zealand which inhibits cytochrome P450 activity is paclobutrazol (Burden et aI 1987), a plant growth regulator used in this country to increase crop yields in stonefruit species (O'Connor 1989). Paclobutrazol inhibits a cytochrome P450 mediated oxidation of kaurene to kaurenoic acid, which therefore prevents production of the plant growth hormone gibberellic acid (Burden et aI 1987).

Field trials were conducted in 1990 by DowElanco staff to determine whether the addition of paclobutrazol to 2,4-D would improve the control of phenoxy-resistant nodding thistle. Despite several application rates being used, no improvement in control of the nodding thistle plants was obtained (B. Harris, pers comm). However application rates ranged from 0.05 -DAD kg ai/ha, yet effects on plants are often not measurable until rates exceed 0.5 kg ai/ha (Hebblethwaite 1987). Therefore this trial did not conclusively show that paclobutrazol is ineffective at overcoming the

resistance mechanism. Gressel (1990) has discussed how there are probably many different types of monooxygenases involved with inhibiting metabolism of herbicides. Thus, even if paclobutrazol is ineffective at overcoming the resistance mechanism, monooxygenases may still be involved and inhibition may be possible using other chemicals. Gressel listed several other chemicals which have been used to inhibit monooxygenases in plants, and any of these may be effective at eliminating resistance in nodding thistle.

Although it would be worthwhile testing these chemicals, there may be practical problems with using them with 2,4-D to improve control of nodding thistle in New Zealand. If a chemical were to prevent deactivation of 2,4-D by nodding thistle, it may also prevent such deactivation in white clover or even grass species, thus increasing damage to desirable pasture species. The addition of another chemical to 2,4-D might also increase the cost of the herbicide treatment substantially, making control of nodding thistle uneconomic.

9.6 CONCLUSION

The mechanism by which resistant nodding thistle plants survived applications of phenoxy herbicides did not involve reduced foliar penetration nor increased exudation from the roots. There appeared to be a small but significant increase in binding of 2,4-D within susceptible plants. However the main mechanism of resistance apparently involved deactivation of herbicide molecules before damage to the plant

could occur. This may have involved hydroxylation of the phenyl ring or

decarboxylation of the side-chain, followed by formation of sugar conjugates, or increased rates of amino acid conjugation or sugar ester formation. Although the identity of the resulting compounds was not established, significant differences were detected between resistant and susceptible plants in radiolabelled metabolites. It may be possible to block this deactivation of 2,4-D by adding a chemical which inhibits the enzymes involved with this metabolism.

CHAPTER 1 0: CONCLUDING DISCUSSION

10. 1 INTRODUCTIOn

As stated in Section 1 . 1 , the objective of this project was to gain an understanding of

why MCPA and 2,4-D consistently give poor control of nodding thistle in parts of Hawkes Bay. It was considered that an understanding of this phenomenon might lead to improved recommendations for the control of this weed.

The development of herbicide resistance within some nodding thistle populations was soon found to be the cause of control problems (Chapters 3 and 4). My discovery of resistance to MCPA and 2,4-D within nodding thistle populations was one of the first documented cases in the world of resistance developing to phenoxy herbicides. The relationship between previous spraying history and the incidence of phenoxy herbicide resistance shown in Chapter 6 gave better proof of resistance having developed as a result of selection pressure from the herbicides than previously documented cases. Other experiments conducted as part of this study showed that cross-resistance occurred to MCPA, MCPB and 2,4-D, but not other "hormone herbicides" such as mecoprop, clopyralid or picloram (Chapter 5). Phenoxy-resistant nodding thistle populations were located at numerous other sites throughout Hawkes Bay and Waikato (Chapter 6). No fitness differential was found between resistant and susceptible biotypes for vegetative growth under nutrient stress (Chapter 7). Physiological investigations showed resistance was due to differences in metabolism between biotypes rather than to differences in herbicide absorption or translocation (Chapters 8

and 9).

Each experiment provided more information about resistance, and implications of the findings were discussed in the appropriate chapters dealing with each experiment. However it would be useful now to draw these findings together in a final discussion of herbicide resistant nodding thistle populations and options available for their control.

10.2 REASONS FOR RESISTANCE DEVELOPING