Genetically engineered bacteria as Biopesticides biopesticides wat r dey? How r dey diff. frm conventional pesticides?

Adv of using biopesticides disadv. Types? Frm whr r dey derived? How do dey function? How can dey b produced? (focus frm bacteria) also give details on the GE bacteria as biopesticides

Biopesticides

According to the United States Environmental Protection Agency (EPA), "biopesticides" are naturally occurring substances (biochemical pesticides) that control pests, microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material, plant-incorporated protectants.

Biopesticides are biochemical pesticides that are naturally occurring substances that control pests by nontoxic mechanisms, derived from such natural materials as animals, plants, bacteria, and certain
minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides. At the end of 2001, there were approximately 195 registered biopesticide active ingredients and 780 products.

Conventional pesticides, by contrast, are generally synthetic materials that directly kill or inactivate the pests. For example, a plant in the presence of chitosan will naturally induce systemic resistance (ISR) to allow the plant to defend itself against disease, pathogens and pests.Biopesticides are considered eco-friendly and easy to use.

The term biological pesticide or biopesticide is used for microbial biological pest control agents that are applied in a similar manner to chemical pesticides. Commonly these are bacterial, but there are also examples of control agents based on fungi, viruses and nematodes.Weeds and rodents have also been controlled with microbial agents.BT-cotton is best example in India. One well-known insecticide example is Bacillus thuringiensis, a bacterial disease of Lepidoptera, Coleoptera and Diptera. Because it has little effect on other organisms, it is considered more environmentally friendly than synthetic pesticides. The toxin from Bacillus thuringiensis (Bt toxin) has been incorporated directly into plants through the use of genetic engineering. Other

microbial control agents include products based on: entomopathogenic fungi (e.g.Beauveria bassiana, Lecanicillium spp.,Metarhizium spp.), plant disease control agents: include Trichoderma spp. And Ampelomyces quisqualis; Bacillus subtilis is also used to control plant pathogens.

Beneficial nematodes attacking insect (e.g. Steinernema feltiae) or Phasmarhabditis hermaphrodita) pests entomopathogenic viruses (e.g.. Cydia pomonella granulovirus).

slug (e.g.

Biopesticides are used to reduce the load of synthetic chemical products being used to control plant diseases. In most cropping systems, biological pesticides should not necessarily be viewed as wholesale replacements for chemical control of plant pests and diseases, but rather as a growing category of efficacious supplements that can be used as rotation agents to retard the onset of resistance to chemical pesticides and improve sustainability. In organic cropping systems, biopesticides can represent valuable tools that further supplement the rich collection of cultural practices that ensure against crop loss to diseases.

Biopesticides fall into three major classes: (1) Microbial pesticides- consist of a microorganism (e.g., a bacterium, fungus, virus or protozoan) as the active ingredient. Microbial pesticides can control many different kinds of pests, although each separate active ingredient is relatively specific for its target pest[s].

Microbial or biological pesticides may consist of bacteria, entomopathogenic fungi or viruses (and sometimes includes the metabolites that bacteria or fungi produce).
For example, there are fungi that control certain weeds, and other fungi that kill specific insects.

The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt. Each strain of this bacterium produces a different mix of proteins, and specifically kills one or a few related species of insect larvae. While some Bt's control moth larvae found on plants, other Bt's are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the particular Bt produces a protein that can bind to a larval gut receptor, thereby causing the insect larvae to starve. (2) Plant-Incorporated-Protectants (PIPs)- are pesticidal substances that plants produce from genetic material that has been added to the plant. For example, scientists can take the gene for the Bt pesticidal protein, and introduce the gene into the plant's own genetic material. Then the plant, instead of the Bt bacterium, manufactures the substance that destroys the pest.

Entomopathogenic nematodesare also often classed as microbial pesticides, even though they are*Plant-incorporated protectants, (PIPs)have genetic material from other species incorporated into their genetic material (i.e. GM crops).
(3) Biochemical pesticides- are naturally occurring substances that control pests by non-toxic mechanisms. Conventional pesticides, by contrast, are generally synthetic materials that directly kill or inactivate the pest. Biochemical pesticides include substances, such as insect sex pheromones, that interfere with mating, as well as various scented plant extracts that attract insect pests to traps.

Various naturally-occurring materials, including fungal or plant extracts, have been described as biopesticides[4]. Products in this category have included: Chitin Chitosan Spinosad Insect pheromones and other semiochemicals

Biopesticides are certain natural plant products that belong to the so-called secondary metabolites, which include thousands of alkaloids, terpenoids, phenolics and minor secondary chemicals. Biopesticides are derived from such natural materials as animals, plants, bacteria, and certain minerals. Biopesticides have usually no known function in photosynthesis, growth or other basic aspects of plant physiology; however, their biological activity against insect pests, nematodes, fungi and other organisms is well documented. Every plant species has developed an built in unique chemical complex structure that protects it from pests.

Perceived advantages
do not leave harmful residues
Biopesticides are usually inherently less toxic than conventional pesticides.

substantially reduced impact on non-target species

Biopesticides generally affect only the target pest and closely related organisms, in contrast to broad spectrum, conventional pesticides that may affect organisms as different as birds, insects, and mammals.

when locally produced, may be cheaper than chemical pesticides in the long-term may be more effective than chemical pesticides Biopesticides often are effective in very small quantities and often decompose quickly, thereby resulting in lower exposures and largely avoiding the pollution problems caused by conventional pesticides.

When used as a component of Integrated Pest Management (IPM) programs, biopesticides can greatly decrease the use of conventional pesticides, while crop yields remain high. To use biopesticides effectively, however, users need to know a great deal about managing pests.

[edit]Perceived

disadvantages

high specificity, which will require an exact identification of the pest/pathogen and may require multiple pesticides to be used often slow speed of action (thus making them unsuitable if a pest outbreak is an immediate threat to a crop) often variable efficacy due to the influences of various biotic and abiotic factors (since biopesticides are usually living organisms, which bring about pest/pathogen control by multiplying within the target insect pest/pathogen) living organisms evolve and increase their resistance to biological, chemical, physical or any other form of control. Unless the target population is completely exterminated or is rendered incapable of reproduction, the surviving population will inevitably acquire a tolerance of whatever pressures are brought to bear - this results in an evolutionary arms

race.

Genetically modified bacteria in agriculture

Abstract Certain bacteria isolated from soils possess properties that allow them to exert beneficial effects on plants either by enhancing crop nutrition or by reducing damages caused by pathogens or pests. Some of them, such as rhizobia, azospirilla, and agrobacteria, have been traditionally released in fields as seed inoculants and they often lead to increases in the yield of different crops while the application of others, such as pseudomonads, often fails to give the expected results. the modified inoculant bacteria after their introduction in field ecosystems effect the resident microflora. Local environmental factors play a crucial role in the survival and persistence of bacteria once released in fields and in the expression of the beneficial traits whether improved or not. The spread of inoculant bacteria from their point of dissemination is limited. Transient shifts in favour of the released bacteria and in disfavour of some members of the bacterial and fungal populations present in the plant rhizosphere might occur with certain released bacteria. The changes observed were, however, less important than those observed under usual agricultural practices. Gene transfer from resident population to introduced bacteria was detected in one case. The transconjugants were found only transiently in the phytosphere of plants but not in soils. No differences between the survival, spread, persistence in field and ecological impacts of genetically modified bacteria and of the corresponding unmodified parent strain could be detected.

1. Introduction
The soil microflora, constituted of bacteria, fungi and algae, plays a key role in the transformations that the principal constitutive elements of living matter undergo within soils. It is responsible for the evolution of numerous organic or mineral compounds, whether endogenous or applied, upon which depend the nutrition and sanitary status of plants . About 108109 bacteria belonging to 103 to 104 species and 104106 fungi are normally detected per gram of ploughed soil. Various bacteria isolated from this huge reservoir of diversity have been identified as being able to achieve functions of agricultural interest such as stimulation of plant growth, protection of plants from pathogens, or the ability to degrade xenobiotics. When applied to soil as crop inoculant, a few of them proved beneficial to the crop and their inoculation with seeds has become a significant agricultural practice. Since they represented an alternative to the use of chemical fertilisers or pesticides, the research developed to identify bacterial mechanisms and genes involved in the production of plant beneficial effects has offered the possibility to improve the expression of useful traits and/or to combine several of them into a single bacterium. 2. Agricultural applications of bacteria Microorganisms can exert beneficial effects on plants directly by enhancing crop nutrition or indirectly by reducing damages caused to plants by pathogens, pests and frost. The first category of microorganisms includes symbiotic or free-living dinitrogen fixers, mycorrhizal fungi, phosphate solubilisers and bacteria stimulating root developmentthrough the production of compounds such as phytohormones, thereby improving mineral and water uptake. The second group is composed of bacteria and fungi able to control soil-borne pathogens,parasitic nematodes or insect pests, competitors of ice-nucleating bacteria and fungal herbicides. 2.1. Bacteria enhancing crop nutrition 2.1.1. Rhizobia Legume inoculation was to improve legume productivity by providing, at planting time, the leguminous plant with its specific rhizobia. These symbiotic nitrogen-fixing bacteria are hosted in nodules that they induce on the roots of legumes and they allow the plant to utilise atmospheric nitrogen. The best example is given by soybean, a legume that originated in China and whose cultivation has been extended to the rest of the world in less than 50 years due to inoculation with rhizobia. For indigenous legume crops, inoculation leads to substantial yield increases as long as it is applied to soils that do not harbour in sufficient abundance indigenous populations of rhizobia specific and effective for the crop.

2.1.2. Plant-associated bacteria Traditional application of bacteria in agriculture is given by inoculation with Azospirillum spp.These bacteria are nitrogenfixing bacteria living in close association with plants in the rhizosphere. They have been shown to promote the growth of various agriculturally important crops in different soils and climatic regions with a 6070% occurrence of success giving increases in yield of 5 30% [22]. The mechanisms of growth promotion is related to the production of growth promoting substances by stimulating the density and length of root hairs and root surface area, would improve the utilisation of water and mineral nutrients. 2.2. Biocontrol bacteria 2.2.1. Control of insect pests Bacillus thuringiensis (Bt) has been the most widely utilised biopesticide for the control of plant damages caused by insects [These sporeforming Gram-positive bacteria produce, during their stationary phase, a large crystal inclusion composed of proteins, the -endotoxins or Cry proteins, which have a larvicide activity on different insect species, characterised by a narrow specificity. This specificity, which allows the preservation of non-target insects and makes the preparations non-toxic for mammals, bird or fish, is one of the advantages presented by Bt preparations over chemical pesticides, another one being their lower capacity to induce resistance. Bt preparations are very unstable under natural environment and present a narrow host range. To overcome these problems, approaches that have been taken include the construction of strains with enlarged host range or increased activity and, with the aim of delivering the Bt toxin in the vicinity of the targeted insect, the introduction of Bt toxin genes into plant-associated bacteria or directly into the plant genome. Bt toxin genes have been introduced in a number of plant-associated bacteria such as root colonisers, Azospirillum[27], Pseudomonas spp. [24], [28], [29] and[30], symbiotic bacteria, Rhizobium leguminosarum[31], or endophytic bacteria, Herbaspirillum seropedicae[24] and Clavibacter xyli subsp. cynodontis[32]. With the exception of this latter species used in field releases with corn [33], the recombinant bacteria have been used mainly to produce Bt endotoxin preparations free of Bt spore[25] and [34]. 2.2.2. Control of plant diseases Root-associated bacteria with the ability to protect plants against a range of soil-borne plant pathogens belonged to the genus Pseudomonas, but bacteria belonging to other genera,Bacillus, Agrobacterium, Enterobacter, Erwinia, Streptomyces, Serratia, Burkholderia. The mechanisms recognised as being responsible for disease suppression in biocontrol bacteria comprise production of metabolites with antimicrobial activity or chitinolytic enzymes. In a given biocontrol bacterial strain, more than one mechanism may function. The relative importance of a particular mechanism may vary with the environmental conditions that prevail in the rhizosphere. With the aim of extending their biocontrol capacity, a number of strains ofPseudomonas have been modified. They include strains that overproduce antifungal compounds, produce Bt endotoxin, or act on biocontrol activity by indirect mechanisms. 3. Survival and spread of modified bacteria introduced in the field environment 3.1. Monitoring inoculant bacteria Monitoring inoculant bacteria after their release in field soils required differentiation of the inoculant bacteria from the many indigenous bacteria that can be genetically and/or phenotypically very similar. The introduction of gene markers into inoculant bacteria, normally uncommon in the soil population,facilitates monitoring of the released bacteria, particularly when they were introduced into antibiotic resistant derivatives, which has been usually the case. 3.2. Survival and persistence 3.2.1. Rhizobia Cultivated soils often harbour populations of rhizobia specific of the commonly grown leguminous crops, either because the leguminous plants are native of the area or as a result of previous inoculations]. However, their levels vary greatly with the soil nature and the host specificity of the considered population. To express their potentialities, genetically improved rhizobia will need to occupy host nodules, hence, following their release in soil, they will have to compete successfully for the formation of nodules with the homologous rhizobia already present in soil. 3.2.2. Pseudomonads The different genetically modified derivatives and parent strains of Pseudomonas inoculated to seed behaved essentially in the same way after their release in the field. Inoculant strains rapidly colonised the crop plant rhizosphere and then declined gradually during the plantgrowing season.

4. Genetic interactions between genetically modified bacteria and indigenous bacteria Evidences that intragenic and intergenic gene transfers occur among soil bacteria have been provided indirectly by population studies and directly in microcosm experiments or in the field environment under selective pressure [85]. The presence of plasmids, genetic elements that replicate independent of the chromosome, is a common feature among soil and plant-associated bacteria. Since many of these plasmids are self-transmissible (conjugative) and can mobilise other plasmids, plasmid transfer is thought to represent the major means of transmission of genetic information among soil and plant bacteria. To investigate as to what extent such transfers occur in the field environment and may participate in gene flux between introduced and resident bacteria, genetically marked bacteria able to act as donors or recipients of mobile genetic elements were introduced as seed inoculants in field experiments and the presence of transconjugants was searched for. These different field releases of strains specifically designed for assessing the transfer of plasmids under natural conditions have provided evidence that, under certain circumstances and with certain bacteria, genes could be transferred from the resident bacterial population to the introduced bacteria. However, as for the numbers, the changes induced in the population are transient at least in the absence of selective pressure on the transferred trait. 5. Efficacy of genetically improved bacteria in field conditions 5.1. Agrobacteria Biological control of crown gall formation, a disease causes by the soil-borne Agrobacterium tumefaciens, has been practised on a very large scale and for more than two decades by using a non-tumorigenic strain of Agrobacterium, A. radiobacter K84 [88]. This strain produces a bacteriocin, agrocin 84, to which A. tumefaciens strains are susceptible [52]. The production of agrocin 84 and the resistance capacity being encoded by plasmid-borne genes, a plasmid transfer event, might cause pathogenicA. tumefaciens to acquire these traits. To prevent such an event, a part of the transfer region was removed from the plasmid. The resulting genetically modified strain was as effective, if not more, than the parent strain [89] and was considered safer in controlling crown gall [90]. In Australia, this derivative has been commercialised in place of its parent strain since 1989 [42]. 5.2. Rhizobia Genetic modifications that have been introduced in rhizobia aimed at improving the strains used in inoculant on traits supposed to be at the origin of inoculant failures: the ability to compete successfully with the indigenous strains for the formation of nodules and the capacity to fix nitrogen. In order to increase the nodulation competitiveness of inoculant strains under field conditions, two different approaches have been taken. One relies on the introduction of genes coding for an antibiotic, trifolitoxin, to which indigenous bacteria are sensitive [15]. It has been used by Robleto et al. [21] who compared the nodulationcompetitiveness of nearly isogenic inoculum strains of R. etli, the common bean microsymbiont, that differed only by their capacity to produce trifolitoxin and were coinoculated to field-growing common beans. They showed that, over 2 years, the trifolitoxin-producing strains occupied at least 20% more nodules than the nontrifolitoxin-producing strains. The other approach is based on a modification of expression of a metabolic gene, putA, involved in the processes of root surface colonisation [91]. Using this approach, van Dillewijn et al. [11] showed that when field-growing alfalfa was inoculated with an S. meliloti overexpressing putA gene, the inoculant strain occupied, for the first month following inoculation, a larger proportion of the nodules than when inoculated with the control strain. Increasing the competitiveness of inoculant strains by genetic modification appears therefore as an accessible goal. Nevertheless, when the crop yield was measured, which was the case in the 3 years of experimentation with common bean, the yield of the inoculated plants did not differ from the yield of uninoculated plants. This implies that the inoculant strain and the indigenous strains were equally efficient in their nitrogen-fixing capacity. Increasing nodule occupancy by the inoculant strain will only benefit the plant when the indigenous competing population is less efficient in nitrogen fixation than the inoculant strain, a case that may not be frequent. 5.3. Pseudomonads The main objectives of the field releases that have been performed so far with genetically modified Pseudomonas strains have been to monitor the fate of marked bacteriaas to whether they improved or not in terms of their biocontrol properties once introduced in field ecosystem and to study the ecological impact of these introductions. We have seen above that the marker genes utilised did not alter the ecological fitness of the pseudomonads in which they were introduced and that the recovery based on these marker genes, was as effective as the recovery based on traditional selection using antibiotic resistance alone. The ecological behaviour of the modified bacteriawas essentially the same as that of wild type bacteria. In a field release aimed at comparing the effectiveness of a lacZY-marked strain and of the parent strain in biocontrol of take-all of wheat, neither the parent nor the marked strain provided control of the disease [41], [42], [43], [44] and [45]. The lack of biocontrol by a strain that had provided effective biocontrol in previous experiments in other locations was attributed to local environment conditions. This points out the difficulties that may be encountered when testing improved strains in natural ecosystems. 6. Conclusion

The disseminations of bacteria in the field have remained marginal, with the exception of rhizobia and agrobacteria, likely because they were not systematically associated with visible beneficial effects. If we want to make greater use of natural or improved bacteria in agriculture, a better knowledge of the factors that are at the origin of these inconsistent performances is needed. By permitting monitoring of the bacteria inoculated to plants in field ecosystems, the first releases of genetically marked bacteriahave allowed some progresses to be made in this direction. Local environmental conditions have appeared as the main factors that regulate successful introduction ofbacteria in the ecosystem and expression of the potential benefits of introduced bacteria. Further identification of these factors will necessitate more field-testing of modified strains over different sites and several years. The experience gained with the field releases conducted so far with genetically modified bacteria has shown that spread from the site of their release was limited and that the ecological modifications in the resident microflora that might be induced were apparently transient and less pronounced than modifications under traditional agricultural practices. Additional experimental field releases of modified bacteria, aimed at determining the factors that influence the expression of beneficial traits in field ecosystem and at testing performances of improved strains, could thus be performed at minimal risks. By allowing definition of the area of potentially successful use and identification of traits to be modified for enhancing crop yield, they should lead to reasoned applications of the selected or modified bacteria, and likely to improved performances of these bacteria in field ecosystems