Sep 15, 2008

Research Projects

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Development of resistance to Bt maize among Western corn rootworm

(2005 – 2008) University of Göttingen, Institute of Plant Pathology and Plant Protection, Göttingen

Topic

This project looked at two questions:

Resistance development on alternative host plants

Bt maize (Cry3Bb1) with resistance to the Western corn rootworm (Diabrotica virgifera virgifera) is only fatal to the first larval stage of the pest. Unlike the European corn borer, the Western corn rootworm can also use other plants which are common in maize fields as a source of nutrition – so-called alternative hosts. This means that in the longer term there is a possibility that partially resistant pests may emerge, which could lead over several generations to full resistance.

The collected data were fed into a model that will estimate the probability of resistance developing as a function of the presence of alternative hosts in the Bt maize.

Refuge design

To slow down the development of resistance, ‘refuges’ of conventional maize are planted. The idea behind them is that the pests can reproduce in the conventional plants, so partial resistances will be thinned out again. The project investigated the role that different layouts of transgenic and non-transgenic plants play in an effective refuge strategy.

Impact on mycotoxin levels

Bt maize varieties that are resistant to the European corn borer are known to have significantly reduced mycotoxin levels. The aim of the project is to investigate whether this is also true of diabrotica-resistant maize.

Summary

Resistance development on alternative host plants

Alternative host plants in Bt maize crops lead to an increased chance of survival for the Western corn rootworm larvae. Both the plant species and the layout within the crop are important.

Refuge design

Beim Vergleich verschiedener Anordnungen von Refugien zeigte sich, dass die “refuge in a bag”- Strategie, bei der dem Saatgut zwanzig Prozent konventionelles Saatgut beigemischt werden, eine Bildung von Teilresistenzen eher fördert als verhindert. Werden Bt- und konventioneller Mais nebeneinander angebaut, sollten mindestens zwei Reihen Abstand eingehalten werden, um ein Überwechseln der im Boden sehr mobilen Larven zu verhindern.

Impact on mycotoxin levels

The presence of the larvae led to a higher infestation with a mycotoxin-producing fungus (F. verticillioides). Where larvae were present in the Bt maize there was fungus as well.

Mycotoxins (Fumonisin) were not detected. However, they are usually produced only when the plants mature. It is therefore suspected that mycotoxins would have been detected at a later sampling date.

Experiment description

Resistance development on alternative host plants

Since the Western corn rootworm is still a quarantine pest in Europe, the studies of resistance development were carried out in the laboratory or greenhouse.

Larval mobility: The mobility of the larvae was tested in two different soil types (soil and sand mixture). This involved planting a maize plant at one end of a long, narrow trough (1m x 10 cm x 10 cm). Five days later larvae were released at the other end. After two, four and eight hours, the larvae were extracted from soil samples at intervals of 10 cm. In this way it was possible to measure the distance covered by individual larvae.

Bioassay: In a bioassay, various plants (grass weeds frequently found in and around maize fields and various cereals) were tested for their suitability as alternative host plants for the Western corn rootworm. Roots of these plants were fed to the larvae and then their weight gain and grazing capacity were measured.

Effect of different age classes of Bt maize plants: In order to investigate the effect of roots of different ages on L2 larvae, 120 Western corn rootworm eggs ready to hatch were placed on two and a half, four, six and eight-week-old Bt maize and isogenic plants. After twenty days, the larvae were extracted from the root balls. The larval stage, head capsule width and dry weight were recorded.

Container experiments: Refuge-design experiments: In order to assess the effectiveness of refuges, the project studied four variants to see how many surviving larvae can be expected in each. First of all, 100% Bt maize was compared with 100% isogenic maize (= refuge). In addition, a row of Bt maize and a row of isogenic maize were used to simulate the interface between Bt and conventional maize, and a seed mix (= ‘refuge in a bag’) was used in which 20% of the plants were isogenic. The larval development was compared.

Influence on the occurrence of mycotoxin The relationship between pest damage to the roots and the mycotoxin contamination was quantified using a bioassay.

Bt-maize and isogenic lines were cultured in a greenhouse. A mycotoxin-producing fungus (Fusarium verticillioides) was introduced into the soil in three concentrations. Eighty corn borer eggs were also added to each plant. After 3 weeks the roots were examined for their colonisation by the fungus.

Results

Resistance development on alternative host plants

Larval mobility: Larval mobility was studied to assess how far the larvae can move from the maize. L2 larvae were found throughout the entire trough and were therefore much more mobile than L3 larvae, which tended to be found close to where they were placed. Larvae in the soil/standard potting soil mixture were significantly more mobile than those in the sand mixture. The soil/standard potting soil mixture was therefore used for subsequent experiments. Artificial compression of the soil, on the other hand, produced no significant differences.

Bioassay: A majority of the larvae (>50%) demonstrated weight gain, in particular with wild oats (Avena fatua), yellow foxtail (Setaria glauca) and winter wheat. How much was eaten by the larvae (grazing capacity) was largely dependent on the individual host plants. For example, for a similar larval weight gain, a great deal of yellow foxtail but only a relatively small amount of wild oats was consumed.

Effect of different age classes of Bt maize plants: The largest numbers of larvae were found on the older plants. As expected, many more larvae were found on the plants of the isogenic line than on the Bt maize plants. Some surviving larvae were found on the Bt maize. Most of these were in the second larval stage on eight-week-old plants. By contrast, on eight-week-old plants of the isogenic line, most of the larvae were already at the third larval stage.

Container experiments: Effect of alternative host plants: The alternative host plants were placed in between two maize rows or within the rows themselves. Planting winter wheat or foxtail millet between the Bt maize rows did not result in higher numbers of surviving larvae than when only Bt maize was available. Where the host plants were planted within the rows, however, significantly more larvae were found. Where rye was used, more larvae were observed in both variants than in 100% Bt maize. However, the largest numbers of larvae were not found on the roots of the alternative host plants, but on the root balls of the Bt maize plants. This ties in with the hypothesis that freshly hatched L1 larvae feed on alternative host plants first and then switch to maize. In these experiments too, a delay in the development of the larvae was observed. In the experiments with the isogenic line most of the larvae were already at the L3 stage, while most of the larvae found in the Bt maize were L2 larvae.

Container experiments: Refuge design: Larvae were found even in 100% Bt maize, but in much smaller numbers than in the isogenic line. In addition, nearly all the larvae observed were L1 larvae, while most of those found in the isogenic line were L2 and L3 larvae.

In the interface variant with 50% isogenic line, as many L1 and L2 larvae were found on the Bt plants as on the isogenic plants. It was only L3 larvae that were significantly less common in the Bt maize.

The ‘refuge in a bag’ strategy with 20% isogenic plants resulted in significantly more L2 larvae than in the 100% Bt maize, but overall much fewer than in the 100% isogenic line. More L2 and L3 larvae were also found on the Bt maize plants themselves than in the 100% Bt maize. Since the L2 and L3 larvae of the Western corn rootworm survive on Bt maize, this strategy tends to promote rather than hinder the formation of partial resistance.

Effect on mycotoxin levels: The presence of larvae led to higher infestation with the fungus F. verticillioides. Even Bt maize had a much higher fungal infestation if larvae were present – despite suffering less root damage. Mycotoxins (Fumonisin) were not detected. However, since mycotoxins are not usually formed until the plants mature, it is likely that they would have been detected at a later sampling date.