CSE1304 set out to test assumptions for refuges; tested if tolerance in addition to resistance could be a potential threat to Bt cotton efficacy and looked at ways to improve refuge governance. This project has identified some issues with refuge assumptions, and increased understanding of tolerance that will be presented to the TIMS technical panel for discussion.

While some assumptions for refuges such as moths from Bt and non-Bt refuges readily mate, and the population is not segregated were found to hold, others did not. It was found that much higher numbers of both Helicoverpa species are emerging from Bt cotton than expected. This suggests that 50% of all moths in Bt/refuge complex (ie excluding unstructured refuges) are originating from Bt cotton. This highlights the importance of both a healthy and attractive refuge, but also growing a healthy Bt cotton crop.

Susceptible Helicoverpa, especially in latter instars, could survive in Bt cotton by feeding on plant structures with low levels of toxin. While H.armigera may be more likely to develop resistance to Cry2Ab, H.punctigera may be more likely to develop tolerance to Cry1Ac. H.punctigera exposed to low levels of Cry1Ac toxins in later instars produced offspring with higher tolerance to Cry1Ac toxin; and those emerging from Bt cotton had higher tolerance to Cry1Ac. Exposure to both Cry1Ac and Cry2Ab concurrently lead to an increase in tolerance levels.

Some planned genetic studies of tolerant colonies were not able to proceed because it was identified that Cry2Ab colony had the HaR01 Cry2Ab resistance gene. It is likely the original 2011 susceptible colony had an undetectable level of the gene that increased in concentration with exposure to low level toxins. Tolerance may assist the survival of RS individuals, thereby assisting the spread of resistance, but this needs more investigation. Tolerance was found to decrease over generations when larvae are no longer exposed to toxins, so refuges may help to reduce the impact of both tolerance and resistance.

Narrow refuges did not detract from the attractiveness of pigeon pea, and attractive pigeon pea did not increase egg lays in neighbouring Bt cotton. Given the mobility of, and the ability of late instar pupae to survive and build tolerance (particularly H.punctigera tolerance to low levels of Cry1Ac toxin) , it was identified that larvae moving from non-Bt plants to Bt (eg deteriorating refuges to Bt cotton) was a threat. Recommendation to have Bt cotton and refuges separated will be raised with the Bt tech panel.

Pigeon pea attractiveness was found to be higher at the end of the season, meaning these refuges may act more like trap crops. The case for destruction of pigeon pea crops at the end of the season will be raised with Bt tech panel.

While satellite imagery is not useful in identifying field attractiveness at a particular point in time, it does indicate season-long vitality and shows potential to check for problematic refuges remotely, although more work would be needed to fully develop techniques.

Moths of the genus Helicoverpa are the most destructive pests in Australian cotton. They have been also some of the most difficult to manage because H. armigera (in particular) has quickly developed resistance (within 5-8 years) to nearly every insecticide used in its control (Whitehouse et al. 2007). To hinder H.armigera developing resistance to Bt cotton, a Resistance Management Plan (RMP) was put in place when Bt cotton was first used commercially in Australia in 1996. As this was over 15 years ago, the RMP has been successful. Nevertheless, in light of the development of resistance to Bt cotton by H. armigera in other parts of the world (Tay et al. 2013) it is important to remain vigilant and keep testing the tools used in the RMP.

A key tool of the RMP is the use of refuges. Refuges help maintain the potency of Bt cotton by producing unselected Helicoverpa moths that mate with any resistant moths emerging from the Bt crop, thereby diluting their genetic contribution to the next generation and slowing the development of resistance.

Refuge governance is based on models with assumptions that are difficult to test on farms. The Helicoverpa Genome Project has mapped all of Helicoverpa’s genes, making it easier to test two assumptions on the frequency of resistant (R) and susceptible (S) genes, and on the degree to which moths mix both within valleys and between Bt cotton and its refuges. If these assumptions are incorrect, then refuges may be underperforming.

Although refuges are designed to counter Bt resistance developing from genetic mutations, a recent CRDC project (03UA002) showed that under laboratory conditions, the exposure of Helicoverpa to low, non-lethal doses of Bt toxins over 12 generations can cause H.armigera to develop inducible tolerance to Bt toxins, to the extent that they are not killed by levels of Bt toxin fatal to susceptible H.armigera. As stressed Bt cotton plants may produce less toxin, and some parts of the plant produce low levels of toxin, inducible tolerance could be another pathway by which Helicoverpa could survive on Bt cotton. An aim of this project is to test the likelihood that inducible tolerance could occur in field crops of Bt cotton, and if so, if refuges could reduce that risk.

For refuges to counter genetic resistance and inducible tolerance to Bt toxins, they must be working optimally on farms and produce as many moths as possible. To do so refuges need to attract sufficient egglays, and then support as many of the resulting Helicoverpa larvae as possible until maturity. For many growers it isn’t clear if their refuges are countering the development of resistance; how to improve the productivity of their refuges; or how to measure the effectiveness of their refuges in order to improve efficacy. Monitoring refuge productivity is a challenge, with current reporting often at odds with on farm realities. A remote method of checking refuges could be used to identify refuges facing difficulties, which could be then ground-truthed. The ultimate aim of this work is to incorporate best management practises into myBMP to improve refuge governance and also to develop better monitoring techniques to identify under-performing refuges which may need more assistance.

The overall aim of this project is to improve the ability of refuges to counter both the threat of resistance developing via genetic mutation, and the potential threat of crop failure via inducible tolerance. By accessing and countering these threats while concurrently developing better refuge management and benchmarking techniques to improve refuge governance, the ultimate aim is to avoid the cost of losing Bt cotton efficacy.