Resistance monitoring of Heliothis virescens to pyramided cotton varieties with a hydrateable, artificial cotton leaf bioassay

Proof of concept was demonstrated for a practical, off-the-shelf bioassay to monitor for tobacco budworm resistance to pyramided Bt cotton using plant filtrates. The bioassay was based on a previously described feeding disruption test using hydrateable artificial diet containing a blue indicator dye, a diagnostic dose of insecticide and novel assay architecture. Using neonate larvae from a Bt-susceptible, laboratory reared tobacco budworm strain, a diagnostic dose for Bollgard II and WideStrike cotton was obtained that limited neonate blue fecal production to 0-2 pellets in 24 h (Bt-resistant larvae produced >2 fecal pellets). The bioassay was tested with three different field populations of tobacco budworm collected from tobacco in central North Carolina (USA) and shown to accurately diagnose susceptibility to Bt. The diagnostic doses were also successfully evaluated with two Bt-resistant, laboratory reared tobacco budworm strains. Shelf life studies showed the assay could be stored for at least 6 months at room temperature (longer storage times were not studied). The application of the bioassay as an easy to use monitoring tool is discussed.     

Roadside surveys support predictions of negligible naturalisation potential for cotton (Gossypium hirsutum) in north-east Australia

A survey of cotton (Gossypium hirsutum) volunteers was conducted along the 1400 km of a cottonseed transport route in north‐east Australia to determine the extent of naturalisation from spilt seed. In each year, 1% of the road distance was inspected for volunteer plants. The survey results were juxtaposed with the eco‐climatic suitability predictions from inferential modelling. Over 3 years, 22 cotton plants were found at eight sites. Within the cotton production region, volunteers averaged 2.28 plants km⁻¹ of road; their most likely source was seed cotton spilt during the previous harvest. Further north, three plants were found over 3 years, at an average density of 0.089 plants km⁻¹ of road; all three plants were found in locations with a positive Ecoclimatic Index. No secondary spread was detected. Roadside slashing reduced plant survival and the potential to produce seed. In the wet tropics, weed competition and slashing prevented volunteers establishing. The surveys indicate that roadsides in north‐east Australia are a hostile environment for the establishment of cotton plants, with no evidence of naturalisation or secondary spread. Current transport practices utilizing fully covered loads present negligible risk of producing naturalised populations of cotton (either transgenic or non‐transgenic) on roadsides in north‐east Australia.     

Effect of Bt cotton expressing Cry1Ac and Cry2Ab, non-Bt cotton and starvation on survival and development of Trichoplusia ni (Lepidoptera: Noctuidae)

Effects of Bollgard II cotton containing two Bacillus thuringensis var. kurstaki Berliner (Bt) toxin proteins (Cry1Ac and Cry2Ab), non-Bt cotton (DPL 491) and starvation on survival and development of cabbage looper, Trichoplusia ni (Hübner), were determined in the laboratory. Larvae of the first four larval instars died when they fed on the terminal leaves of Bt cotton plants at 50 days after planting (DAP). However, 51.3% of fifth instars that fed on 50 DAP Bt cotton leaves pupated, and 87.1% of the pupae successfully developed into adults. Of the unfed fifth instars (starved), 55.6% pupated and 88.1% of the pupae emerged. Pupae that developed from larvae fed on Bt cotton leaves and unfed were significantly smaller, being 89.7 and 73.2% of the weight of the pupae that developed from larvae fed on non-Bt cotton leaves. Leaves of 120 DAP Bt cotton were less toxic to T. ni larvae. When the first instars continuously fed on 120 DAP Bt cotton leaves, 75.9, 60.6, 56.4 and 38.4% of larvae survived to second, third, fourth and fifth instars respectively, and 20.9% pupated and 17.9% successfully became adults. However, it took the surviving first instars 37.1 days to become adults, which was 7.2 and 8.9 days longer than those fed on 50 and 120 DAP non-Bt cottons respectively. Pupae that developed from larvae that fed on 120 DAP Bt cotton leaves were only 50.9 and 52.6% of the weight of those developed from larvae that fed on 50 and 120 DAP non-Bt cotton respectively. Non-Bt cotton, both 50 and 120 DAP, did not exhibit significant effects on larval survival and development, except that the pupae in the 50 DAP non-Bt cotton treatments developed over a significantly longer time than those in the 120 DAP non-Bt cotton treatment.     

The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC₅₀ and LC_99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC₅₀ and LC_99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour.