GENETICALLY ENGINEERED BIOPESTICIDES

DIVISION OF ENTOMOLOGY

GENETIC ENGINEERING:

• Genetic engineering is defined as direct manipulation of an organism’s genome using biotechnology.

• It is a set of technologies used to change the genetic makeup of cells including transfer of geneswithin and across species to produce improved and novel organisms

BIOPESTICIDES• Biopesticides are certain types of pesticides

derived from such natural materials as plant, animal ,bacteria ,and certain minerals

• Biopesticides are inherently less toxic than conventional pesticides

• Biopesticides generally affect only target pest• Biopesticides are effective in very small

quantity and often decompose quickly

GENETICALLY ENGINEERED BIOPESTICIDES

The engineered biopesticides may include

• Fungi• Bacteria• Protozoa • Viruses

FUNGI• Various fungi such as Beauveria bassiana and

Metarhizium anisoplie have attractive characteristic for insect control

• One major limitation of use of entomopathogenic fungi is that their effectiveness depends on relative humidity

• Need for genetic engineering arises to overcome this and increase their insecticidal efficacy

• M. anisoplie normally takes 5 to 10 days to kill insect .in order to increase its insecticidal efficacy, multiple copies of pr 1 gene were introduced

• This gene encodes subtilisin like protease which helps in host cuticle penetration

• Pr 1 was produced by recombinant fungus in the hoemocoel of lepidopterous larva

• Infection resulted in partial hydrolysis of haemolymph protiens and extensive melanisation of larva

• Larva died from these effects 25% sooner than wild types

• Feeding damage was reduced by 40%

BACTERIA

• There are numerous entomopathogenic bacteria but few have been engineered for enhanced insecticidal properties for use in biocontrol

• Most attention has been given for increasing insecticidal property of Bacillus thurengenesis and bt toxins

• Bacillus thurengenesis is gram positive spore forming bacteria

• The insecticidal property of Bacillus thurengenesis is due to insectecidal cry protiens[ICP]

• ICPs are encoded by cry genes located on extrachromosomal plasmid

• The genes are classified according to their specificity into cryІ to cry ѵґ

–Two limitations of BT are

• relatively lower persistance under field conditions

• the dessemination of large no. of spores which is percieved to be problem in some countries in which products with viable bt spores are not authorized

–To over come these problems following strategies were adopted:

1. ICPs were introduced into nonpathogenic strain of Pseudomonas flourescence

Bacteria were killed resulting in encapsulated ICPs that has enhanced residual property in field and no bt spores

2. Bt δ endotoxins have been engineered into chromosome of pseudomonas flourescens .This was helpful for increasing the range of habitats that can be exploited for bt based insect pest control

3.The gene encoding cry 1Ac has been engineered into clavibacter xyli

The engineered bacterium was introduced into corn seedlings by wound or seed inoculation

Tunneling damage caused by European corn borer ostrinia nubinalis was reduced by 55 to 65%

• Therefore for bt, genetic engineering can be used to increase the activity of particular strain against target pest species,extend host range ,increase field stability,and provide alternative constructs to facilitate management of resistance

Protozoa• The majority of protozoa for insect pest

control are in phylum microspora • In theory genetic engineering could be

applied to microsporidia to enhance host range and pathogenecity

• In practice ,the fact that the microsporidia infect all vertebrate classes in addition to invertebrate classes raises concerns over genetic manipulation

VIRUSES

• The only insect viruses that have been engineered are the baculoviruses

• Genetic engineering has been applied to enhancement of baculo virus since 1980 and has resulted in products that approach the efficacy of chemical insecticides

CONSTRUCTION OF RECOMBINANT BACULOVIRUS

• The most common approach for construction of recombinant baculovirus involves cotransfection of an insect cell line that supports viral replication with naked viral genome DNA And a plasmid transfer vector

• The transfer vector consists of viral genomic restriction fragment containing the desired alteration

• Homologous recombination between the parental virus genome and the transfer vector on either side of alteration (a double crossover event) results in the incorporation of the alteration into viral genome

• Because naked baculovirus DNA Is infectious when introduced into a permissive cell line,the transfection leads to production of progeny virus

• Clonal isolates of recombinant virus are identified and isolated in a plaque assay

RECOMBINANT BACULOVIRUS INSECTICIDES

• Approximately 20 recombinant baculoviruses have been engineered for improved insecticidal efficacy

• The viruses expressing neurotoxins are considered to have greatest potential for commercialization with reduction in time for virus to take effect of upto 60% compared to those of larva infected with wild type viruses

The requirements for insecticidal peptides or protiens expressed in recombinant baculovirus include

1. The protein should have no effect on the ability of virus to infect or replicate in host insect cells before death of host.

2. The protein should be insect specific.

3. Protein should be active at low dosage.

4. Protein should have rapid effect on host .

ORIGIN OF TOXIN

TOXIN VIRUS NAME

REDUCTION IN ET50

LEPIDOPTERAN HOST TESTED

Pyemotes tritici(straw itch mite)

TXP 1 Vsp-Tox34Vp6.9tox34HzEGTDA-26tox34

40%60%40%

Trichoplusia Trichoplusia H. Zea

Androctonus australis(north african scorpion)

AaIT Acst 3AcAaITBmAaIT

10% TO 38% T. niHeliothis virescens

Leiurus quinquestriatus(scorpion)

LqhT1LqhIT2LqhiT3

AcLIT1.p10AcLIT2.p01AcLqhIT3

24%32%20%

O. nubilalisH.armigeraB.mori

ORIGIN OF TOXIN

TOXIN VIRUS NAME

REDUCTION IN ET5O

LEPIDOPTERAN HOST TESTED

Anemonia sulcata(sea anemone)

As II vSAt2p+ 38% T.ni

Agelenopsis sulcata(american funnel web spiner)

µ-Aga-ґѵ vMAg4p+ 37% Spodoptera frugiperaT. ni

Diguetia canites(primitive weaving spider

DTX9.2 vAcDTX9.2 9 TO 24% T.niH.virescens