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The aim of this study was to test the potential of proteinase inhibitors to controlChrysomela tremulae, a beetle that causes severe damage in young plantations and in short-rotation intensive culture (SRIC) of poplar. As a first step, cysteine proteinases were determined to be the major digestive proteinases ofC. tremulae and oryzacystatin OCI, a cysteine proteinase inhibitor, was shown to inhibit this activityin vitro. The gene encoding OCI was introduced into poplar (Populus tremula ×P. tremuloides) and transgenic plants expressing OCI at a high level were selected. Feeding tests on these transgenic plants demonstrate the toxicity of OCI-producing poplar leaves againstC. tremulae larvae.

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6,400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D LC = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D ML = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.

The effect of wastewater irrigation on resistance of poplar trees to the cottonwood leaf beetle, Chrysomela scripta F., was investigated. Five Populus clones were tested in a greenhouse for their effects on the survival, fresh pupal weight, and development time (from egg to adult) of the cottonwood leaf beetle when they were irrigated with either wastewater or freshwater. Foliar concentrations of nitrogen, mineral elements (P, Mn, Mg, Ca, Na, K), phenolic glycosides, and leaf toughness also were measured to understand the potential mechanisms of resistance to the cottonwood leaf beetle. C. scripta performance was negatively affected by irrigation with wastewater; both survival and pupal weight were reduced. Performance also varied among clones and was the worst on Populus fremontii Watson variety wislenzensii, a native species. Leaf toughness and foliar N level were not affected by the irrigation treatment and were not correlated with larval growth and survival. Among minerals, foliar concentrations of sodium increased with wastewater irrigation, and were related negatively to pupal weights. Foliar magnesium also was influenced by the irrigation treatment but it was not correlated with C. scripta performance. Manganese varied with clone, but not with the type of water used for irrigation, and was correlated negatively with survival and related positively to development time. Differences in phenolic glycosides were observed between treatments and among clones, but there were no detectable relationships between phenolic glycosides and C. scripta performance.