K. S. Powell

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

Transgenic potato plants with enhanced resistance to the peach-potato aphid Myzus persicae

Potato plants (Solanum tuberosum) cv. Desireé were transformed with the genes encoding the proteins bean chitinase (BCH), snowdrop lectin (GNA) and wheat α‐amylase inhibitor (WAI) under the control of the constitutive CaMV 35S promoter. Transgenic plants with detectable levels of foreign RNA were then selected for further characterisation with respect to protein expression levels by immunodot blot analysis using polyclonal antibodies raised against the respective protein. With the exception of WAI, plants expressing high levels of RNA, expressed correspondingly high levels of the foreign protein (1.5–2.0% of the total soluble protein). Although high levels of WAI mRNA were detected in some of the transformants, the protein could not be detected. On the bases of expression levels, two lines, designated PWG6#85 (transformed with the double construct WAI/GNA) and PBG6#47 (transformed with the double construct BCH/GNA), were selected for testing in aphid trials for enhanced levels of resistance.

Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, Nilaparvata lugens (Stal).

Rice brown planthoppers (Nilaparvata lugens) were fed on artificial diet containing snowdrop lectin (Galanthus nivalis agglutinin; GNA), which has been shown to be toxic towards this insect pest. In addition to decreasing survival, the lectin affected development, reducing the growth rate of nymphs by approximately 50% when present at a concentration of 5.3&mgr;M. Immunolocalisation studies showed that lectin binding was concentrated on the luminal surface of the midgut epithelial cells within the planthopper, suggesting that GNA binds to cell surface carbohydrate moieties in the gut. Immunolabelling at a lower level was also observed in the fat bodies, the ovarioles, and throughout the haemolymph. These observations suggest that GNA is able to cross the midgut epithelial barrier, and pass into the insects circulatory system, resulting in a systemic toxic effect. Electron microscope studies showed morphological changes in the midgut region of planthoppers fed on a toxic dose of GNA, with disruption of the microvilli brush border region. No significant proteolytic degradation of GNA was observed either in the gut or honeydew of planthoppers fed on lectin-containing diet. The presence of glycoproteins which bind GNA in the gut of the brown planthopper was confirmed using digoxigen-labeled lectins to probe blots of extracted gut polypeptides.

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.

Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens

The feeding activity of adult rice brown planthopper, Nilaparvata lugens (Stal) on selected proteins incorporated into artificial diets was examined, based on quantitative analysis of honeydew excretion, in order to determine the mechanism of action of antimetabolic proteins towards homopteran pests. The lectins Galanthus nivalis agglutinin (GNA), wheat germ agglutinin (WGA) and the enzyme soybean lipoxygenase (LPO), previously demonstrated to be toxic to this insect, reduced honeydew excretion levels of adult Nilaparvata lugens over a 24 h period when incorporated into artificial diet at 0.1% {w:v}, indicating that these proteins acted as antifeedants. Of the proteins tested GNA was the most effective antifeedant, reducing honeydew droplet production by 96%, although after 24 h there was some recovery in the honeydew excretion levels and thus the insects appeared to tolerate the presence of the antifeedant with time. The lectin Pisum sativum agglutinin (PSA), previously demonstrated to be non‐toxic, showed no antifeedant properties.

Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens

Insect feeding trials were carried out to determine the effects of a range of mannose‐specific lectins on third instar nymphs of the rice brown planthopper (BPH), Nilaparvata lugens. Stål. Dose response curves show that Galanthus nivalis agglutinin (GNA) has the strongest toxic effect of the lectins tested, and is effective at concentrations considerably lower than those previously reported. Narcissus pseudonarcissus agglutinin (NPA) and Allium sativum agglutinin (ASA) exhibit a significant antimetabolic effect towards the insect but were less effective (on a molar basis) than GNA. LC50 values for GNA, NPA and ASA are approximately 4 μM, 11 μM and >40 μM respectively. These mannose‐specific lectins are serologically identical, but differ in the number of subunits per protein molecule; ASA is a dimer, NPA is a trimer and GNA is a tetramer. The results obtained support the hypothesis, that the effectiveness of the mannose‐binding lectins as antimetabolites is determined by the number of subunits per molecule. Two N‐acetylglucosamine binding lectins, the dimeric Oryza sativa agglutinin (OSA) and the monomeric Urtica dioica agglutinin (UDA), were also tested but at a concentration of 0.1% w/v exhibited no significant antimetabolic effect towards BPH, although the related lectin wheatgerm agglutinin (WGA) has previously been demonstrated to be toxic towards the insect.

Potential of plant-derived genes in the genetic manipulation of crops for insect resistance

The plant kingdom provides a rich and diverse source of secondary compounds. A protective role against various pests, pathogens and competitors for many of these has been established and in recent years the utilisation of such compounds in crop protection, either by conventional plant breeding or by genetic engineering has been, and is being, investigated (Gatehouse et al., 1990).

Antimetabolic effects of plant lectins towards nymphal stages of the planthoppers Tarophagous proserpina and Nilaparvata lugens

Taro Colocasia esculenta (L. Schott) and rice (Oryza sativa L.) form a major part of the staple diet of pacific islanders. Pest constraints hamper the sustainability of taro and rice production in the Pacific region. Insect feeding trials were conducted in vitro to determine the effects of plant lectins against planthopper pests of taro and rice. Lectins were incorporated into artificial diet at 0.1% (w/v) level. The lectins Galanthus nivalis agglutinin (GNA) and concanavalin A (Con A) showed significant antimetabolic effects towards third instar nymphs of taro planthopper (Tarophagous proserpina Kirkaldy) whilst Pisum sativum agglutinin (PSA) showed no significant effects toward the insect. Psophocarpus tetragonolobus agglutinin (PTA) showed significant antimetabolic effects towards third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål). PTA also reduced honeydew excretion levels of rice brown planthopper, over a 24‐hour period, demonstrating antifeedant properties of the protein.

Yield decline of sweet potato in the humid lowlands of Papua New Guinea

Sweet potato (Ipomoca batatas (L,) Lam) is the major staple crop in Papua New Guinea and experiments were conducted investigating factors affecting yield decline. Yields; of unfertilized plots were related to rainfall and measured changes in soil properties, nematode (Meloidogyne sp., Rotylenchulus reniformis) and sweet potato weevil (Cylas formicarius) populations. The research took place at two locations (Hobu and Unitech) on Eutropepts and Fluvents, respectively. Yields at Hobu decreased from 18 Mg ha(-1) in the first season to around 7 Mg ha(-1) in the third season, but no significant yield trend was observed at Unitech. Vine biomass was not affected by the number of cropping seasons at Hobu brit it decreased at Unitech with time. Marketable tuber yield at both sites was significantly correlated to rainfall, i.e, the more rain the lower the yield. Significant changes in soil chemical properties included a decrease in pH and base saturation (Hobu) and a decrease in CEC and exchangeable K (Unitech). No significant changes in soil bulk density were found and no obvious pattern was found in the nutrient concentrations of leaf samples with time. Nematode populations were high and tripled between the first and third season at Hobu. Half of the vines at Hobu and all of the vines at Unitech were damaged by sweet potato weevils, but tuber damage was higher in Hobu although the damage was only superficial. Despite the considerable variation in yield and yield determining factors, the study showed that the decline in sweet potato yield may be attributed to the high nematode infestation, accompanied by an increase in vine damage by weevils and a declining soil fertility

Phylloxera infested grapevines have reduced chlorophyll and increased photoprotective pigment content – can leaf pigment composition aid pest detection?

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a root-feeding pest of grapevines. In Australia, phylloxera-infested vineyards are subjected to quarantine restrictions and early detection remains vital for the timely implementation of post-outbreak quarantine protocols. Current detection methods rely on time-consuming ground surveying, which involves detailed examination of grapevine (Vitis vinifera L.) root systems. Leaf pigment composition is often a sensitive indicator of plant stress. The increasing popularity of remote sensing systems, which exploit those changes in pigments observed with plant stress, offers a real possibility for the development of a phylloxera-specific remote detection system. Our objective was to investigate changes in grapevine leaf pigments associated with phylloxera infestation and to relate any changes to appropriate reflectance indices. This was achieved with a glasshouse experiment in which the responses of two vine cultivars (Cabernet Sauvignon and Shiraz) to phylloxera infestation were compared with their responses to water and nitrogen deficiencies. The responses of leaf pigments to phylloxera infestation were also investigated in Pinot Noir and Cabernet Sauvignon grapevines grown under field conditions. A reduction in the leaf chlorophyll content and an increase in photoprotective pigment concentrations were observed in leaves of phylloxera-infested grapevines compared with uninfested vines. The photochemical reflectance index (PRI) was found to be most closely associated with the ratio of total carotenoid to chlorophyll in these vines.