Research in Resistance Management

Evidence of the shift in susceptibility to Bacillus thuringiensis delta-endotoxin CryIAc in Australian Helicoverpa armigera (Lepidoptera: Noctuidae)

Ho.T Dang
Australian Cotton Research Institute
NSW Agriculture
Wee Waa Road
Narrabri
Australia
hod@mv.pi.csiro.au

Robin Gunning
NSW Agriculture
Calala Lane
Tamworth
Australia
robin.gunning@agric.nsw.gov.au

INTRODUCTION H. armigera and H. punctigera are important pests of cotton in Australia. Transgenic cotton (producing CryIAc protein) has been commercially grown in Australia for six years. The susceptibility of both species to Bacillus thuringiensis (Bt) toxins in Australian field populations has been monitored since 1993.

Three Bt commercial products containing different proteins have been used for testing the survivorship of Helicoverpa spps larvae, namely Dipel(R), Xentari(R), and MVP(R) (Table 1). The results of monitoring against Dipel(R) and Xentari(R) have not been changed to date. However, the survivorship of a Helicoverpa spps larval tested against MVP (contains CryIAc, Table 1) significantly increased during the early part of the 2000/01 cotton season, especially H. armigera.

This paper reports the results of Bt resistance monitoring to date and evidence of the shift in the susceptibility with possible esterase mediated mechanism of resistance in H. armigera.

MATERIALS AND METHODS

1. Bioassays in resistance monitoring program
Helicoverpa eggs were collected throughout the season in cotton and other summer crops in cotton growing regions in Australia. Eggs were then transferred onto an artificial diet, hatched, and maintained to early third instar larvae. Larvae were then, transferred onto a "testing diet" into which a discriminating dose of the Bt product had been incorporated (Fig 1). Dipel(R), Xentari(R), and MVP(R) were used for screening (Table 1). Larval mortality was assessed after seven and ten days of exposure to the testing diet, respectively. The discriminating dose for each product was the LC99 for tested species. Survivorship of larvae on diet incorporated with the discriminating dose of Bt products was monitored.

In collaboration with Monsanto Australia, mortality was assessed using larvae hatched from the over wintering pupal populations collected in August, 2001.

2. Preliminary study on genetics of resistance
Since February, 2001, the survival of field populations H. armigera was found to be significantly increased. Experiments were carried out to investigate the possible mode of inheritance of the resistant trait.

H. armigera larvae surviving the discriminating dose of MVP (3ul/ml diet) were removed from the "testing diet" and maintained on the feeding diet (without MVP) until pupation. Surviving moths were bred to produce selected resistant strains (called Silver). Probit analysis was run on bioassay results of various generations of the Silver strain in comparison with that of the susceptible laboratory strain and field non-selected strain.

Silver F4 female moths were used for crossing with male moths of the susceptible strain (KO) to produce the hybrid strain (Silko F1). Silko F1 was bioassayed in comparison with other strains.

3. Assessment of resistance on whole plant and in field trial

4 Resistance mechanism studies
The Envirologix test kit was used to conduct a preliminary analysis for the presence of toxin CryIAc in surviving and dead larval from the MVP screen. The result revealed that dead larvae contained higher levels of CryIAc than surviving larvae indicating that there was difference in the rate of detoxification in the individuals Subsequently, we investigated the difference in esterase activity in susceptible and Silver F2 strain.

Homogenates from mid-gut of susceptible (KO) and Silver F2 H. armigera larvae were incubated with concentrations of purified CryIAc. Total esterase activity was detected using 1-naphthyl acetate as a substrate. Incubates was then run on polyacrylamide gels and stained for esterase activity.

RESULTS AND DISCUSSIONS

1. Result of Bt resistance monitoring program
The percentage survival of Helicoverpa armigera and H. punctigera against the discriminating dose for each product form 1996/97 to 2000/2001 seasons is presented in Table 2. Monitoring for Dipel(R) resistance has been conducted since 1996 and there has been no change in susceptibility of field populations to this product. A similar result was obtained for Xentari(R). On the other hand, survival of both species to MVP(R), which contains the single Bt toxin (CryIAc), increased significantly. Average survival of H. armigera against MVP(R) for the crop season 1999/2000 and 2000/01 was 2.6% and 7.1% respectively. Bioassays of H.armigera field collections conducted in August, 2001 by Monsanto across six cotton growing regions indicated that the survival had increased to 9.2% (average of six regions - Table 3)

The differences in survival for H. armigera against MVP(R) and Dipel(R) in the two years 1999/2000 and 2000/01 are shown in Fig 1 and 2.

A closer examination of the increase in survival of H. armigera during 2000/01 (Fig. 1), indicates a significant increase in H. armigera survival in February and March. In later months of the crop season, the survivorship declined. The extent of H. armigera's survival appears to be related to the to the level of Bt expression in transgenic cotton throughout the season.

2. Inheritance of resistance in selected strain
Table 4 shows the LC50, LC99 and resistance factor of different Silver generations (selected strains), field, susceptible strains, and the cross between susceptible and selected strain (SilkoF1).

The resistance factor increased as the strains being selected and reduced when crossed with susceptible strain. The resistant trait proved to be heritable characteristic.

3. Larval survival on whole plant and in field trial
Table 5 shows the survivorship from neonates to pupae of Silver F4 (selected strain) and KO (susceptible strain) on caged plants under greenhouse conditions. Larvae of KO strain died after the first week of exposure, as compared to 10.75% of larvae of Silver F4 strain which survived through to pupation.

In the field trial, the larval mortality of field collected strain on single gene Bt transgenic varieties (average of four varieties) was lower than that on two-gene variety (Fig. 3). The mortality was corrected using mortality on non-Bt conventional variety. It is apparent that the single gene transgenic varieties had reduced efficacy against the H. armigera field collected insects.

Leaf samples assessed for CryIAc content (Envirologix Test Kit) indicated that CryIAc was present at high concentration until the plants were about 100 days old. However, mortality of insects was very low even at the start of the season. At the peak of Bt expression in single gene transgenic varieties (at 48 and 54 days after seeding -DAS), there was 64% and 53% reduction in efficacy as compared to two-gene variety. If the field collected strain was susceptible to CryIAc, such reduction in efficacy would not be expected. Thus, the field strain used in this experiment which was collected in October/November, 2000 might have been less susceptible to CryIAc.

4. Resistance mechanism - Esterase sequestration in resistant strain
Polyacrylamide gels developed from this study showed that the selected resistant strain had greatly increased esterase activity (Fig 4) and there were considerable differences in esterase banding patterns between these strains. Data in Fig 5 show that CryIAc inhibited esterase activity in the resistant strain, where as there was no detectable binding of CryIAc to esterase in the susceptible strain.

Esterases are enzymes in H. armigera and other insect pests, which detoxify many insecticides by hydrolysis and sequestration. Sequestration by esterases has been characterised as the primary cause of pyrethrod resistance in Australian H. armigera. Preliminary research by Dr R. Gunning and Dr. G Moores (IACR, Rithamsted, UK), has shown that activity of esterase in the gut of the Bt resistant H. armigera strain binds readily for the CryIAc pro-toxin. Given the greatly increased esterase activity in the resistant strain, it is likely that considerable amount of CryIAc pro-toxin could be sequestered before reaching the target site. Our preliminary data in comparing the extent of CryIAc sequestration in various H. armigera strains showed that levels of sequestration are decreasing in the following order (1) Silver F2 (resistant strain), (2) Emerald (field strain), (3) SF4 x KO (cross between resistant and susceptible strain) and KO(susceptible strain). Similar order is also observed for the resistance factor of these strains (Table 4) , Thus, the level of esterase sequestration is closely related to the resistance factor found in probit analysis.

SUMMARY Ingard(R) cotton provides a valuable tool for the management of Helicoverpa spps in the Australian cotton industry. Maintaining susceptibility of insect populations to Bt proteins as well as to new chemistries is essential for the industries sustainability. The actual impact of efficacy of transgenic crops due to development of resistance is difficult to measure. The current change in H. armigera susceptibility would not be detected by commercial field checks for Helicoverpa infestations. Also, any loss of efficacy in transgenic crops would be difficult to establish as the majority of transgenic cotton crops in Australia are regularly sprayed with larvicides after flowering. The use of non-Bt conventional insecticides in transgenic crops would mask the reduced efficacy.

The evidence of the shift in susceptibility demonstrated in our studies might have impact on the field performance of one gene transgenic varieties. Further research focussing on the assessment of field performance of Ingard cotton would be required to ascertain this possibility. Such study will determine not only the impact of the shift in susceptibility on the field efficacy of the Bt toxin in one gene transgenic crop but also effect on the efficacy of the future two-gene transgenic cotton. While the mechanisms of Bt resistance in Helicoverpa field populations are not well understood, enzymatic sequestration of toxin recognised by Bt research workers as a potential resistance mechanism. In the case of H. armigera, it is likely that considerable amount of CryAc pro-toxin is being sequestered. Esterase sequestration however might be only one of a number of mechanisms involved in Bt resistance.

A number of the assumptions that was made in the development of the current resistance management strategy for Bt cotton have proved to be unsubstantiated (Daly and Olsen, 2000; Tabashnik et al, 2000). Improving our understanding of the mechanisms and genetics of resistance to CryIAc and new Bt toxins is seen as a priority in the development of future resistance management strategy.

ACKNOWLEDGMENTS: We are thankful to the Cotton Research and Development Corporation (CRDC-Australia) for funding this research project. Thanks to Dr Neil Forrester (Deltapine International) and Mr Philip Armitage (Cotton Grower Service) and Mr Stewart Addison (Monsanto Australia) for their assistance in various studies. Many thanks to all members of the team including Mrs J.Chapman, Mrs C. Dang, Mrs D. Richardson and Mrs K. Stanford for the bioassays.

REFERENCES

Daly, Joanne and K. Olsen. 2000. Genetics of Bt resistance. In: Proceedings of the 10th Australian Cotton Conference, Brisbane, Australia ,August 2000.

Gillespie, P. 1995. Probit 5 for window P38. In: proceedings of the 26th AGM and Scientific conference of the Australian Entomological Society , Tamworth, Australia 24th -28th September ,1995.

Tabashnik, B.E., A.L. Patin, T.J. Dennehy, Yong-Biao L, M.A. Sims and L. Antilla 2000. Frequency of resistance to Bacillus thuringiensis in field populations of Pink bollworm. Proceedings of the National Academy of Sciences of the US of America PP: 12980 - 12984.