Insect and pest resistance

Tobacco Growing Made Easy

How To Grow Tobacco At Home

Get Instant Access

Several strategies to control insects and pests in fruit crops are currently used. Control by insecticides is the most used, followed by biological control, use of resistant plants and other integrated insect pest control techniques. The massive use of pesticides, beyond environmental pollution, induces insecticide resistance among the insect populations, leading to new insecticides and their increased use. Resistance is controlled by partially recessive or co-dominant (additive) genes and involves a small number of loci. Resistant plants in fruit crops are rare, and their development is difficult. Genetic engineering offers new approaches to more rapid deployment of anti-insect strategies in fruit crops (Table 3.6).

Resistance to Bt protein has been studied with the aim of retarding the spread of resistance in the insect population and the strategies involve:

• use of high doses of insecticidal protein in order to kill the homozygous resistant insects, driving the insects away from the crop;

• multiple target strategy, aiming to use multiple insecticides;

• the refuge strategy aims to express in plants a dose of insecticide able to kill heterozygous insects and the survival of homozygous ones. The density of the homozygous population can be kept low by planting a mixture of transgenic resistant plants and non-transgenic ones on which susceptible insects could survive and mate with homozygous resistant insects thus creating heterozygous ones (Alstad and Andow 1995).

Several plants have been engineered with the aim of killing phytophagous insects by the following strategies:

1. genes encoding insecticidal crystal protein from Bacillus thuringiensis

2. proteinase inhibitors

3. lectins

4. alpha-amylase inhibitors

5. chitinases

6. polyphenol oxidases and peroxidases

7. lipoxygenases

8. ribosome inactivating proteins (RIPs) and promising insecticidal proteins, isolated from microbial culture filtrates, such as cholesterol oxidase, Vip3A and Tca (Escobar and Dandekar 2000).

Genes encoding insecticidal crystal protein from Bacillus thuringiensis Individual strains of Bacillus thuringiensis have been characterised and classified according to their insecticidal activity (Schnepf et al. 1998). This activity is due to their insecticidal crystal proteins (ICPs) which are toxic for several important insect pests for fruit and nut crops. There are several individual ICPs which are highly specific to a particular insect order, including Lepidoptera, Diptera and Coleoptera. Recently B. thuringiensis (Bt) strains have been isolated with activity against Hymenoptera, Homoptera, Orthoptera, Mallophaga, nematodes, mites and protozoa (Schnepf et al. 1998; Escobar and Dandekar 2000). Since the individual ICPs are released from parasporal crystal in response to alkaline conditions (pH 9-10) in the midgut of the target insect, toxicity may depend on solubilisation of some ICPs and the number and type of receptors in the midgut microvillae of insects (Du et al. 1994; McGauhey and Whalon 1992). Genes encoding these ICPs have been cloned from Bt and are referred to as cry genes (cryIAa, cryIAb, cryIAc). They have been transferred to several plants including fruit crops (Table 3.7). However, in some transgenic plants, such as apple and walnut, the wild type gene sequences encoding the cryIA(c) revealed no expression. Subsequently the gene sequences were restructured and these problems eliminated, giving rise to successful transgenic plants (Dandekar et al. 1994). It has been demonstrated that damage by such transgenic plants to non-target insect populations is less than that caused by chemical pesticides (Losey et al. 1999).

Fruit crop

Alien gene(s)

System/Plasmid or selective agents

Origin of plant material

Insect target

Expressed protein

Authors

Apple (Malus X domestica) cv Greenleaves

CryIAa (c)

A.t.

Leaf segments

Lepidoptera

CryIAc

Dandekar 1991; Dandekar 1992

Apple (Malus X domestica) cv Greenleaves

CpTI; CryIAa

A.t.

Leaf segments

Lepidoptera Coleoptera

CpTI

James et al. 1992; 1993

Cranberry (Vaccinium macrocarpon)

CryIAa

A.t.

Lepidoptera

CrylAa

Singh and Sansavini 1998

Cranberry (Vaccinium macrocarpon) Ait.

Icp

PB

Stem section

-

-

Serres and Stang, 1992

Grape (Vitis vinifera)

CryIAa

A.t.

Lepidoptera

CrylAa

Singh and Sansavini 1998

Grape (Vitis vinifera)

gna

A.t.

Lepidoptera

GNA

Coghlan 1997

Grapefruit (Citrus paradisi Macf.)

gna

A.t.

Aphids

GNA

Yang et al. 2000

Juneberry (Amelanchier laevis)

Btk-icp

A.t.

Basal cut end of shoots

-

Toxin HD73

Hajela et al. 1993

Juneberry (Amelanchier laevis)

cryC

A.t.

Lepidoptera, Coleoptera

Unspecified Ccry gene

Krattiger 1997

Pear (Pyrus communis)

cryC

A.t.

Lepidoptera, Coleoptera

Unspecified Ccry gene

Krattiger 1997

Persimmon (Diospiros kaki) CryIAc

A.t.

Leaf discs

Lepidoptera

CryIAc

Tao et al. 1997

Strawberry

(Fragaria X ananassa)

cpti

A.t.

-

Expressed CPTI

James et al. 1992

Strawberry (Fragaria X ananassa) some cultivars

cpti

A.t.

Vs Otiorhynchus sulcatus

Expressed CPTI

Graham et al. 1996

Walnut (Juglans regia) hybryds & cv Sanland

CryIAc

A.t.

Somatic embryos

Lepidoptera

CryIAc (vs codling moth)

Dandekar et al. 1992; Dandekar 1994, 1998

Proteinase inhibitors

Amino acids are essential for herbivorous insect survival and are metabolised by proteinase in the insect gut. The availability of one amino acid rather than another depends on the pH of the insect gut (Wolfson and Murdock, 1990). In plants, proteinase inhibitors that are highly specific for some classes of insect proteinases are induced in response to mechanical or insect damage, reversibly binding the active site of proteinases and forming an inactive complex (Laskowski et al. 1987; Boulter 1993) which reduces the availability of amino acids necessary for insect nutrition. A potent natural insecticide was found in cowpea and identified in tripsin inhibitor (CpTI), which confers resistance to cowpea seed weevil. Over-expression of this proteinase gene in plants may have an insecticidal activity by also increasing the hyperproduction of proteinases in the insect gut, depleting the insect's metabolic reserve of sulphur-containing amino-acids (Broadway and Duffey 1986). Although several crops have been transformed and the protein has been expressed, insecticidal effect has been reported in a few cases. Since the insects seem to develop a rapid resistance following a continuous ingestion of proteinase inhibitors (Jongsma et al. 1995; Broadway 1995, Girard et al. 1998), the effectiveness of this strategy for plant protection still needs to be established.

Lectins

Lectins are proteins with specific carbohydrate binding activity. Many of the over 300 purified lectins from seeds are toxic for animals and seem to have multiple roles in plant physiology. They are targeted to the vacuoles or secreted extracellularly. Some of them are toxic for Coleopteran, Lepidopteran, Dipteran and Homopteran insects (Van Damme et al. 1998). Considering that the last group of insects, up to now, is not controlled by any other insecticidal protein, lectins have aroused particular interest (Gatehouse and Gatehouse, 1998). An insect diet containing mannose-specific snowdrop lectin (GNA) is effective in reducing larval growth of Coleoptera and in reducing fecundity of adults of peach and potato aphids (Myzus persicae) (Sauvion et al. 1996), while N-acetylglucosamine from castor bean and wheat germ (WGA) is toxic for some Lepidoptera. The mode of action is not clear yet, though it seems to be linked to endocytosis in the intestine (Zhu-Salzman et al. 1998). Up to now, expression of insecticidal lectins in transgenic plants has provided relatively low protection against Lepidopteran and Homopteran pests, and their potential toxicity to mammals limits their use in the growing of fresh fruits.

Alpha-amylase inhibitors

The presence of these proteins in seeds seems to protect them from insect attack by inhibiting midgut-a-amylase, reducing the ability of the insect to catabolise starch (Baker et al. 1991). They were isolated from common bean, named aAI-1 and aAI-2, and are active mainly against Coleopteran a-amylase. Application in fruit crops, by overexpressing them in the tissues, seems at the moment limited, since inhibition of gut-a-amylase may not be an antinutritive deterrent to insects that feed upon leaves or phloem sap and, in addition, could limit the activity of mammalian a-amylase.

Chitinases

Besides their activity against fungal pathogens, chitinases also have potential for insect control, since the exoskeleton and peritrophic gut membrane of insects are constituted by chitin. Up to now, plant chitinases have not shown high insecticidal activity (Kramer and Muthukrishnan, 1997). However, following the studies of Ding et al. (1998), who fed tobacco budworm with transgenic-chitinase tobacco foliage and sub-lethal doses of ICP of B. thuringiensis, transgenic plants overexpressing both chitinase and ICPs seem to provide a promising strategy in insect control. It seems that the degradation of peritrophic gut membrane, operated by chitinase, increases the accessibility of the ICP to epithelian cell membrane receptors, enhancing the insecticidal property (Ding et al. 1998).

Polyphenol oxidases and peroxidases

Both polyphenol oxidases (PPOs) and peroxidases catalyse the oxidation of phenolic compounds to reactive quinones (Steffens et al. 1994), perhaps complimenting each other in a generalised antinutritive response (Duffey and Felton 1991). Following cell lysis, the contact of PPOs or peroxidases with the phenolic substrate produces quinones which can irreversibly degrade nucleophilic amino-acids that are essential to insect diet, limiting herbivorous insect growth. Studies with potato, either overexpressing PPO or reducing it by antisense-PPO, increased or reduced insect mortality respectively (Steffens et al. 1994). Antisense-peroxidases in tobacco plants, however, did not reduce larval susceptibility. Peroxidases also induce rapid lignification response at plant wound sites, which may play a role in insect defence. However, abnormality due to high lignin content and root system mass reduction was evident in some transgenic tobacco plants. Plants overexpressing anionic peroxidases caused in some cases, but not in others, an increase of mortality of Lepidopteran and Coleopteran larvae, including the woody transgenic sweetgum. Both strategies may have a limited application in fruit crops.

Lipoxygenases

Lipoxygenases catalyse degradation (peroxidation) of free unsaturated fatty acids, which are essential in insect diet. Since the activity of plant lipoxygenases has been associated with tissue wounding, their increase in the wounded tissues could represent an active antinutrient strategy for reducing damage caused by herbivorous insects (Felton et al. 1994; Royo et al. 1999), reducing also palatability (Duffey and Stout 1996) and regulating the expression of other wound-responsive defence genes (e.g. through jasmonic acid) from pathogen attack.

Ribosome inactivating proteins (RIPs) and other compounds Studies on these groups of plant proteins are quite recent (Barbieri et al. 1997). RIPs were isolated from microbial culture filtrates. They are similar to compounds such as cholesterol oxidase (Shen et al. 1997), Vip3A (Yu et al. 1997), and Tca (Bowen and Ensign 1998), and show insecticidal properties when added to the insect diet. In addition a new class of molecules named neuropeptides is under observation, they include proctolin, schistocerca allatostatin-5, locustanin 2 (Kelly et al. 1990). The insects exposed to them experienced dismetabolic effects, growth inhibition and death (Tortiglione et al. 1999).

At present only BtlCP transgenics maintain acute toxicity in the absence of other insecticidal proteins (Roush 1998; Escobar and Dandekar 2000). Field trial experiments demonstrated a rapid evolution, similar to chemical insecticides, of resistance to insecticidal proteins such as a-amylase, proteinase inhibitors ICP of B. thuringensis (Michaud 1997).

To maintain long-term insect resistance in transgenic crops, several strategies have been suggested (Roush 1998; Escobar and Dandekar 2000).

• cultivation of multilines containing different insect resistant genes

• transgene pyramiding (multiple insecticidal proteins expressed in a single crop cultivar)

• cultivation of susceptible plants for insect refugia

• expression of the transgene only in specific plant tissues.

Since insects show different levels of resistance, according to the homozygosity, high resistance in insects acquired after consuming tissues of transgenic crops could be reduced by crossing with the large non-resistant insect population colonising non-transgenic refugia crops (Gould 1998). Few fruit crop genotypes have yet been transformed with generally poor results. However, genes such as the ipt gene, under a wound-inducible promoter, have produced insect pest resistance in tobacco plant (Smigocki et al. 1993). Pyramiding transgenics, particularly with BtICP and cholesterol oxidase, seem to be good candidates for future transgene strategies, possibly using inducible or situ-specific promoters.

Was this article helpful?

+1 0

Post a comment