Australian Herbicide Resistance Initiative (AHRI)

Digging deep into 2,4-D resistance

Written by: Cindy Benjamin

Synthetic auxins might be the oldest kids on the herbicide block but that doesn’t mean they are well understood. In fact, there are very large knowledge gaps that researchers like AHRI’s Dr Danica Goggin are trying hard to fill in a bid to find ways to overcome resistance to this herbicide group in weeds such as wild radish.

As recently as 2016, international researchers published a paper explaining how synthetic auxin herbicides like 2,4-D actually work. As outlined in AHRI Insight #64 How stuff works: 2,4-D, free radicals and monkeys, we now know that synthetic auxin herbicides cause the over-expression of genes responsible for cell elongation, cell division and hormone production. Instead of enabling the plant to grow at an extraordinary rate, this overexpression causes excessive production of free radicals, along with excessive ethylene production and closure of the plant’s stomata; both of which also cause additional production of free radicals. This usually results in unusual plant growth and eventual plant death following the application of 2,4-D or dicamba.

Don’t be fooled by the delicate flowers of wild radish! It’s a very problematic weed. Photo by: Danica Goggin.

How does wild radish survive?

In a plant species such as wild radish that is known to be susceptible to this mode of action, how does herbicide resistance evolve?

To find out, Danica originally studied one susceptible and two known resistant populations of wild radish, using radioactive 2,4-D to determine whether resistant and susceptible plants translocated the herbicide differently. The results were stark and Danica thought she had solved the puzzle! It was clear from this experiment that the resistant wild radish plants were able to prevent the herbicide from moving out of the leaves that it was applied to, keeping the vulnerable growing points safe and allowing the rest of the plant to grow as normal and ultimately ‘survive’ the herbicide application. See AHRI Insight #63 2,4-D Gridlock.

Digging deeper proved that the answer is more complex

Having identified a very plausible resistance mechanism in two wild radish populations, Danica went on to test the translocation theory in another nine 2,4-D resistant populations of wild radish from WA.

Danica characterised the resistance profile of the 11 resistant populations to find that resistance to 2,4-D and dicamba appears to be capped. This conclusion is based on results showing that selection with 2,4-D did not increase the level of resistance and that progeny of paired crosses were no more resistant than the parents.

“Charlie, are you having any luck sniffing out these 2,4-D mechanisms?” “Clancy, all snout sensors are on the job –  let’s keep digging!”

Characterisation of the 11 resistant populations also showed that while all 2,4-D resistant populations were also resistant to dicamba, the level of resistance to the two herbicides varied, suggesting that there is no consistent cross-resistance to these two auxinic herbicides within a population.

Conducting the same experiment with the radioactive 2,4-D applied to the leaves, she also found that translocation varied enormously both between and within resistant populations. In fact, several of the resistant populations translocated 2,4-D just as efficiently as susceptible populations.

This proved that wild radish has other, more complex mechanisms at its disposal to avoid the effects of excessive free radical production following exposure to phenoxy herbicides.

On further investigation, Danica demonstrated that there is nothing to indicate that wild radish uses enhanced, irreversible metabolism as a resistance mechanism for 2,4-D. Having ruled this mechanism out, all the evidence is now pointing toward mechanisms involving auxin signalling and plant defence genes.

This diagram shows the biological processes that went up in the Resistant plants but down in the Susceptible plants in response to 2,4-D. Bigger and darker blue indicates increased importance.

Preliminary studies suggest that gene IAA30 may contribute to the ability of a wild radish population to grow in the presence of 2,4-D. The association of this gene with 2,4-D resistance was more clearly observed in the root elongation experiments that were conducted in a controlled (indoor) environment compared to the foliar experiments that were conducted outdoors. This suggests that environmental conditions may also play a part in determining which mechanisms provide the best defence for wild radish populations in different geographic areas. There’s additional evidence that a component of the plant stress–response system is already ‘switched on’ in 2,4-D-resistant plants, even before the herbicide is applied.

Delving right down to cell membrane level

Given the variety of auxin responses within and between the 11 resistant populations, Danica is sure that there is more than one signalling protein involved in 2,4-D resistance in wild radish. Given the high genetic variability found in wild radish populations, there is good reason to suspect that repeated use of 2,4-D could alter the sequence or expression of different co-receptor components in different populations, affecting not only their resistance to various auxins, but also the fitness of the survivors.

This diagram shows groups all the genes that went up in Resistant plants but down in Susceptible plants – the story of resistance.

Resistance is still resistance and requires diverse management tactics

Evolved resistance to 2,4-D in wild radish is now believed to be conferred by a variety of auxin signalling defects that all result in resistance levels of 4–8 times the recommended field rate. While some resistant populations appear to suffer a fitness penalty, this is not always the case.

Further research will investigate strategic mixes of 2,4-D and synergists to help increase the efficacy of 2,4-D in resistant populations. Any herbicide treatments will rely heavily on complementary strong crop competition and harvest weed seed control.

Any synthetic auxin-tolerant corn, soybean and cotton cultivars introduced into Australia will potentially increase selection pressure on 2,4-D and will require careful stewardship to manage herbicide resistance.


You can read more about the paper ‘2,4-D and dicamba resistance mechanisms in wild radish: subtle, complex and population specific?’ and download it here.