Endogenous resistance to insect pests in alfalfa: engineering for enhanced resistance

Abstract

Alfalfa (Medicago sativa) is a valuable forage crop grown throughout the World. While the crop is resistant to attack by many insect pests, it is subject to potentially severe losses through the action of several specific pests, which are adapted to alfalfa as a host. The most economically damaging of these pests is the alfalfa weevil, Hypera postica. This thesis investigates the endogenous defences of alfalfa against insects, which are responsible for its resistance to non-pest species, and develops a strategy for increasing the resistance of alfalfa towards pest species, specifically alfalfa weevil. The role of saponins in the resistance of alfalfa towards non-pest species has been investigated by using successive insect bioassays, carried out with extracts, mixtures of compounds, and purified compounds, to identify which compounds present in alfalfa tissues are responsible for toxicity towards insects. Crude saponin extracts, in 80% methanol, from alfalfa seedling tissues were bioassayed against the cowpea seed weevil, Callosobruchus maculatus. Both extracts from shoot and root tissues caused larval mortality and delayed development when incorporated into an artificial diet at levels comparable to those found in alfalfa, but lower levels of root saponin extracts showed probiotic effects, whereas lower levels of shoot saponins were still toxic. Hydrolysis of the saponins present in these extracts decreased their toxicity. Purified saponin mixtures were prepared by butanol partition and ether precipitation, and were bioassayed against potato aphid (Aulacorthum solani) in a liquid artificial diet, which allowed quantitative effects to be accurately assayed. Shoot saponins showed a concentration-dependent toxic effect, decreasing survival over an initial 5 day period, decreasing growth, and inhibiting fecundity (measured by nymph production) in these insects. Alfalfa root saponins showed no deleterious effects below a threshold level, but caused complete mortality above this level. The alfalfa saponin mixtures were separated into fractions by chromatography on a reverse phase column. Bioassays showed that the toxicity towards potato was associated only with fractions containing saponins, and that fractions containing a component identified as soyasaponin I were more toxic to the aphids than others. Finally, two saponins purified from alfalfa, soyasaponin I and medicoside A, were assayed. These assays showed that soyasaponin was consistently more toxic in effects on mortality, growth and fecundity. It was concluded that alfalfa saponins, and in particular soyasaponin I, were major factors in the resistance of alfalfa towards potato aphid, and other insects. A saponin mixture from another species, sugar beet {Beta vulgaris) was also toxic to aphids, supporting the view that saponins have a general role in resistance to insects. Inhibition of insect digestive proteolysis by expression of a foreign protein protease inhibitor was selected as a strategy to protect transgenic alfalfa against alfalfa weevil. The major protease activity in larvae of this msect was shown to be due to cysteine proteases, which could be inhibited by cystatins. Rice cystatin was produced in large quantity using a recombinant protein expression system in E. coli for use in a "proving" experiment. Incorporation of the rice cystatin into an alfalfa weevil larvae artificial diet decreased survival, showing that this approach was feasible. [brace not closed]