BIOPESTICIDES

Biopesticides include "naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material (plant-incorporated protectants) or PIPs." They control pests by nontoxic mechanisms. The most successful BCA is Bacillus thuringiensis, a spore-forming bacterium containing crystalline protein inclusions. The proteins are highly toxic to insect pests but specific in their activity. They have been widely used for over 30 years against Lepidoptera (caterpillar) pests. The commercial application of these biopesticides is usually as formulations of spores and crystalline inclusions of disrupted B. thuringiensis. In recent years the toxin genes have been isolated and sequenced and recombinant DNAbased products produced and approved. These genes have also been incorporated into various plant species and expressed in the plant tissue. This now offers the farmer either direct application of the microbial biopesticides or protection of the plant directly by toxic gene expression in the plant tissue.

Negative Impacts on the Environment

The pest species with repeated exposure to a certain biopesticide may with the due course of time develop resistance towards these natural toxins. These resistant varieties will no longer be controllable with the biopesticides and further may transmit these genes to other species causing further dilution of genetic identity among insect or plant species. Also the wild type varieties of different pests may pose new environmental and agricultural problems once they incorporate the resistance gene. Effects may be farfetched leading to imbalances in the interspecific interactions and ultimately the natural food cycle and habitat.

Gene Flow from Plant-Incorporated Protectants

The movement of transgenes from the host plant into weeds and other crops has been a significant concern due to the possibility of novel exposures to the pesticidal substance. This concern has been considered for each of the Bt plant-incorporated protectants (PIPs) currently registered. Adverse environmental impacts, include those which may arise from gene flow of PIPs to wild or feral populations of sexually compatible plants. They may enter the food supply whether through seed mixing or cross pollination. Bt Corn, cotton and potato were reviewed for their potential to hybridize with wild and feral relatives of sexually compatible plants in the U.S., its territories and possessions.

The Bt corn and potato have been expressed in agronomic plant species that, do not have a reasonable possibility of passing their traits to wild native plants. Most of the wild species in the United States cannot be pollinated by these crops (corn and potato) due to differences in chromosome number, phenology and habitat. There is a possibility, however, of gene transfer from Bt cotton to wild or feral cotton relatives in Hawaii, Florida, Puerto Rico and the U.S. Virgin Islands.

Ecological Non-Target Organism Risk from Biopesticides:

Hazard to non-target organisms is a function of toxicity and exposure. Non-target organisms that are and organisms that are exposed to limited amounts of a toxic substance may not be at risk. Therefore, the risk assessment process depends on determination of the toxicity of a given substance and the degree of environmental exposure of non-target species. The primary issue which defines the type of toxicity data needed for a risk assessment of PIPs is that the pesticidal substance is contained within the plant parts thus resulting in minimal exposure to non-target organisms. Exposure of non-targets to plant-incorporated protectants would occur primarily when wildlife feed on the pesticidal plants, on exposure to dispersed pollen or if sexual transfer of the new trait(s) to non-target wild/weedy relatives by crosspollination takes place. Therefore, each risk assessment should be made from an analysis of the nature of the gene being introduced, the plant receiving the gene, the environment where the plant will be grown and the species susceptible to the effects of the introduced gene.

Environmental Fate
Fate and Environmental Effects of Transgenic Bacillus thuringiensis d-Endotoxins in the Soil Soil organisms may be exposed to d-endotoxins from current transgenic crops by exposure to roots, incorporation of above ground plant tissues into soil after harvest, or by pollen deposited on the soil. Root exposure may occur by feeding on living or dead roots or, theoretically, by ingestion or absorption after secretion of d-endotoxin into the soil. In addition, evidence suggests that some soil components, e.g. clays and humic acids, bind dendotoxins in a manner that makes them recalcitrant to degradation by soil microorganisms, but without eliminating their insect toxicity. Therefore, exposure to d-endotoxin bound to soil particles may also be a route of exposure for some soil organisms. Experiments addressing the amounts and persistence of d-endotoxins in the soil have been submitted and reviewed. A number of publications have addressed the degradation of Cry proteins in the soil. Cry protein half-life studies were submitted for registration for corn containing Cry1Ab, and published studies were available for Cry1Ac cotton. Cry1Ab produced estimated degradation rates of 1.6 days for the Cry protein as expressed in transgenic corn tissue and 8.3 days for the purified protein.

Several studies indicate that Cry proteins bind to clays and humic acids. The results of these studies suggest that this binding slows the rate of microbial degradation of these toxins compared to when these soil components are not present. However, this protection is not absolute, since degradation does in fact occur under several experimental conditions. Several factors influence either the affinity of binding or the rate of degradation. Exposure of Non-Target Organisms to Cry Proteins in the Soil The route of exposure of non-target organisms must also be considered. In the case of significant root exudation of Cry protein, some differences in exposure compared to single incorporation of transgenic plant material may be expected. Organisms that pass soil through their digestive systems, such as earthworms, could be exposed to higher levels of Cry protein due to exudation compared to a single incorporation of plant material. Organisms at higher trophic levels that feed on soil feeders may, secondarily, be exposed to toxin. Testing on birds and rodents however, showed that there was no toxicity to these species. It is very difficult to determine the importance of shifts in the structure of microbial soil populations unless these changes can be associated with measurable ecological parameters. In most cases, such research has not been performed for microbial populations

Development of Resistance to Biopesticides

Theoretical Basis for Resisitance
There are several differences between the nature and use of a chemical pesticide and a biopesticide which need to be taken under consideration for possibility of development of insect resistance. The nature of the chemical pesticides are novel; for the most part these molecules have been never been encountered by the target insect prior to introduction by man. Chemical agents often kill by interaction with a single host system. Microbials, in contrast, are organisms with which the host insects have had contact for millions of years which may interact with many host systems before causing death. These agents have had limited usage till date. The insecticidal protein toxins of the Bacilli e.g Bacillus thuringiensis, represent possible exceptions to these principles, in that they are essentially chemical insecticides, produced by a microbial agent. They are very complex molecules of great variety with complex modes of action, apparently with a long history of interactions with insects, and unstable environment exposure. Thus they share characteristics with both chemical and microbial pathogens. Other pathogenic microorganisms are very different in their interactions with their hosts from chemical insecticides and the development of resistance to these organisms may be expected to be slower and of a different nature then resistance towards chemicals.

A change in the response of an insect population following selection pressure may be reflected in a reduced proportion of insects which succumb to a given discriminating dose of the pathogen, and increase in the LC50 or LD50. Insect populations, particularly wild populations, consist of a mixture of individuals with varying susceptibility, and elimination of the most susceptible individuals may lead to changed response without the presence of true resistance.

Resistance to Bacillus thuringiensis

A number of studies have already examined the possibility of induction of resistance to B. thuringiensis toxin in target insects. Highly variable levels of control of the stored grain pest, Plodia interpunctella, were found in wild populations from grain bins treated with B. thuringiensis and in laboratory populations. Failure to control the pests in the grain bins treated with B. thuringiensis was later shown to be due to resistance which in some cases developed within a years treatment. Further studies showed 30-fold resistance in 2 generations and 100-fold in 15 generations. As a stored grain pest P. interpunctella encounters microbial pathogens in a almost constant environment, without sunlight load or seasonality experienced by field crop pests. These environmental factors and high genetic heterogeneity of this insect may make it particularly susceptible to the development of resistance. Studies with other organisms showed different results, for e.g almond moth, Cadra cautella showed only 7-fold increase in resistance over 21 generations. The resistance trait appeared to be recessive of partially recessive. A tobacco budworm Heliothis virescens was selected with a B. thuringiensis var. Kurstaki HD1 toxin. Gradual increase in resistance was seen by the seventh generation. One of the most well documented cases of resistance following use of commercial B. thuringiensis products against o field crop pest was demonstrated in the case of the diamond back moth Plutella xylostella. These pests in the farms of Hawaii repeatedly treated with Dipel, were found to be upto 41-fold more resistant then laboratory populations or field populations with minimum exposure.

Insects Resistant To Biopesticides

There are many instances in which insect pest populations have developed resistance to selective (alternative) pesticides or biopesticides. Here are several examples: Green peach aphid (Myzus persicae) resistant to azadirachtin (e.g., Azatin) Western flower thrips (Frankliniella occidentalis) resistant to spinosad (e.g., Conserve)

 Leafminer (Liromyza trifolii) resistant to both spinosad (e.g., Conserve) and abamectin (e.g., Avid) Greenhouse whitefly (Trialeurodes vaporariorum) resistant to buprofezin (e.g., Talus) Sweet potato whitefly B-biotype (Bemisia tabaci) resistant to pyriproxyfen (e.g., Distance) Diamondback moth (Plutella xylostella) resistant to Bacillus thuringiensis spp. kurstaki (Dipel)

Resistance to Biopesticides in Wild Populations

Among wild population of insects fewer cases of resistance to pathogens have been documented, perhaps only because of lack of study. Resistance to infection by granulosis virus was observed in a population of European larch bud moth, Eucosoma griseana, following an epizootic of this virus and similar changes in susceptibility to nuclear polyhydrosis virus occurred in populations of the California oakworm, Phyganidia californica, following an epizootic. This is still one of the most carefully analysed studies of resistance in field population. The authors presented the hypothesis that resistance followed by gradual return to susceptibility, may be responsible for the cyclic occurrence of viral epizootics.