Introduction

There are currently two ways in which an exotic pathogen may be introduced into a country for biological control of a pest. It may enter as a ‘classical’ introduction, to be released and established for continuing control of a (usually exotic) pest, or it may enter as a formulated pesticide, either registered or in an experimental form. Either way, when released in the field, there is a likelihood of its establishment. There is a need, therefore, in both cases, to ensure that such introductions are safe to the environment, and pose acceptable risks to non-target organisms.

Introducing exotic pathogens – the classical precedent

Over the past century, the classical biological control of insect pests and weeds has been dominated by the introduction of insect control agents (see Hopper, Chapter 6; and Blossey, Chapter 8). The use of pathogens in classical biological control has been limited, but it is increasing as we come to understand more about the biology of pathogens and see the positive results from a handful of successful programmes.

Against insects pests, the success of the baculovirus of Oryctes rhinoceros on Pacific and Indian Ocean islands is well-known (Bedford, 1986). More recently, a number of exotic introductions have been made against other insect pests, including some cnew associations’. For instance, the fungus Entomophaga praxibuli has recently been introduced into the US from Australia for grasshopper control (Anon, 1989) and an isolate of Entomophaga grylli has been introduced from Australia to the US against the indigenous grasshopper, Phaulacridium vittatum (Milner, 1985).

Introduction

During the 1970s a set of powerful biochemical tools became available for the characterization and manipulation of nucleic acids and proteins. These included the ability to sequence DNA segments rapidly, to excise specific segments, to execute changes to the nucleotide sequences, if and as desired, and then to insert the modified segment elsewhere into the genome. During this period it was also demonstrated that genetic material could be transferred into the genomes of foreign organisms and, under appropriate conditions, be expressed thus leading to synthesis of RNA and protein products. For example ‘expression systems’ using eukaryote genes coding for proteins such as interferon, insulin, growth regulators, etc. could be constructed in bacteria, yeast, insect or other eukaryote tissue cultures. Expression of these foreign genes in the host represented a valuable source of such products.

In the latter part of the 1970s and for much of the 1980s, recombinant-DNA technology was largely used as a powerful and creative tool in molecular biology and genetic analysis. It was principally directed towards fundamental questions such as the improvement of our understanding of gene structure and function and for unravelling chromosome organization.

The notion that novel genetic combinations could be created with recombinant-DNA (r-DNA) technology, which would have been unlikely or impossible to arise in nature, caused molecular biologists to pause and consider the risks from inadvertent or deliberate construction of novel organisms that might cause environmental or health problems should they enter the environment by accident or design.

Introduction

Biological control by introduction and permanent establishment of exotic natural enemies of pests has been practised for over 100 years. Although a few introductions of beneficial insects had been made earlier, the introduction of an Australian ladybird (Rodolia cardinalis Mulsant) into California to control the cottony cushion scale (Icerya purchasi Maskell) on citrus in 1888–9 (Caltagirone and Doutt, 1989), is generally considered to mark the beginning of the practice of biological control as an effective pest control strategy. This operation was not only highly successful in controlling the pest but also was widely publicized with the result that economic entomologists in a number of countries were soon importing ladybirds for the control of a wide range of pests in what Lounsbury (1940) referred to as the ‘ladybird fantasy’. Few of these introductions were successful, consequently practitioners began to study the ecology and population dynamics of pest natural enemy systems and a more scientific approach to biological control developed (Waage and Greathead, 1988), with a consequent increase in the success rate. However, the introduction of natural enemies remains a largely empirical activity that depends to a large extent on the knowledge and insight of the practitioner.

There are other ways in which natural enemies can be applied as pest control agents; by augmentative or inundative releases of native or exotic agents, which includes formulation of pathogens as biological pesticides (see Huber, Chapter 18; and Waage, Chapter 9) and conservation or enhancement of the action of native species of natural enemies (see Edland, Chapter 4). Because the introduction method was the first to achieve an outstanding success, it is now referred to as ‘classical biological control’.

Introduction

A major attraction of the application of genetic engineering to microbial inoculant strains is the ability to create new strains with capabilities that may not be easily obtained by a process of natural selection. Such additional features may enhance the performance of an inoculant strain.

The fluorescent Pseudomonas species are an important group of bacteria that can play a beneficial role in the protection of crop plants against deleterious pathogenic micro-organisms. These bacteria produce a wide range of factors antagonistic to pathogens (particularly fungi) they include the yellow-green, fluorescent siderophores that characterize this group.

Our work has concentrated on the role of siderophores and antibiotics of fluorescent Pseudomonas in the biocontrol of ‘damping-off’ disease of sugar-beet seedlings (see Table 14.1). The biosynthesis and excretion of iron-binding siderophores are thought to decrease the pool of iron available to pathogenic fungi and other harmful micro-organisms and so contribute to the plant growth promotion effect observed when plants are inoculated with the appropriate Pseudomonas strain (Kloepper et al 1980). The producing micro-organism can use the resulting Fe3+ (ferric) siderophore complex via specific receptors located in its outer membrane (Magazin et at., 1986).

Outer membrane receptor proteins can be highly specific and only transport the appropriate ferric siderophore into the producing cell. The structures of these fluorescent (yellow-green) siderophores was reviewed by Leong (1986). Fluorescent siderophores are only produced under iron-limiting conditions, as are receptor proteins.

Introduction

Host specificity screening is the most important step that each potential weed biological control agent (whether insect or pathogen) has to pass prior to its introduction (Schroeder, 1983). Regardless of its control potential, only the demonstrated safety of plants of economic or ecological importance in the release area, will lead to approval for release of the control agent. The use of screening-protocols for weed biological control agents (Wapshere, 1975; Schroeder, 1983; CAB, 1986) are now widely accepted. Even less rigorous tests in the first part of this century have never led to the release of a ‘new pest’ (Crawley, 1989; Howarth, 1991).

The safety record of weed biocontrol using insects is excellent, nonetheless, the growing awareness of the public towards environmental interference by man, has led to legislative regulations such as the ‘Biological Control Act of 1984’ in Australia. Today, information on the environmental impact, alternatives, the relationship between local short-term uses and long-term productivity, and a risk analysis of the proposed action are required. Conflicts of interest over the implementation or safety of a specific biological control programme or agents, may still arise (Delfosse, 1990).

During the last decades especially, the critical objections of nature conservationists who fear for already stricken and endangered plant species related to the target weed, have considerably delayed the progress of programmes (Schroeder and Goeden, 1986). Despite the excellent safety record of weed biocontrol, we have to assess the risks of introducing a foreign species into a new environment.

Introduction

Biotechnology (genetic engineering) and biological control offer many opportunities to improve agricultural production. Potential benefits include: achieving higher crop and livestock yields; improving nutritional make-up of crops and livestock; substituting biocontrols for pesticide use; as well as controlling some soil and water pollutants. At the same time, the use of biotechnology and release of genetically engineered organisms (for biocontrol and other purposes) into the environment could create serious ecological, social, and economic hazards (Pimentel et al, 1989).

One objective in the implementation of any new technology is to achieve maximum benefits with minimal risks to the environment, economy, and society. The other major objective is to be able to use the technology so that all future benefits are realized. If a serious problem results from the application of just one project, the future development of the entire technology is jeopardized. Note what has happened to nuclear energy in North America because of mismanagement by industry and the government.

In this article, the potential risks of the use of biotechnology and release of genetically engineered crops and biocontrol organisms into the environment are assessed. Various approaches are suggested that could be employed to minimize and reduce the risks of biotechnology to the environment and society.

Ecological issuesCrop resistance to pests

The engineering of crop resistance to insect and plant pathogen pests offers advantages to reduce the use of insecticides and fungicides in crop protection. This will generally reduce problems from pesticides (NAS, 1989; Pimentel et al, 1993) and improve the economics of pest control (Pimentel, 1986).

Introduction

The agro-ecosystems in Fennoscandia are characterized by relatively few and moderate plant protection problems. The number of pests and plant diseases is smaller than in warmer regions. Due to a shorter growing season and lower temperatures, many multivoltine insects develop only one generation a year. The beneficial fauna, primarily the many predaceous bugs (anthocorids and mirids) and mites (phytoseiids), have also proved to be highly efficient as control measures.

Pest control systems have been developed for several agricultural crops, which require minimum inputs of pesticides. The programmes for many greenhouse cultures, based on biocontrol, and those for fruit crops, using integrated approaches, have been especially successful.

Development of IPM in Norwegian fruit orchards

In the 1950s and early 1960s a spray programme that consisted of 6–8 annual applications was commonly used against insects and mites. This heavy routine spray programme was expensive and caused undesirable environmental impact.

An extensive research programme on IPM, which started in the 1960s, provided important information on the biology and ecology of major pests and their natural enemies. Based on this new knowledge, the use of pesticides could be reduced. By 1974, farmers who had adopted IPM in their orchards, used only 0–3 pesticide applications a year without significant damage to the crop (Edland, 1989a). During recent years, common use of scab-warning devices has resulted in further reduction of spray programmes. Thus, in 1988 the average use of insecticides and acaricides was 1.1 sprays and of fungicides 3.5 sprays, for the whole season in many orchards (Edland, 19896). This is less than 20% of what is being used in integrated fruit production in some other European countries (Prinoth, 1990).

Introduction

Many attempts have been made to control soilborne plant pathogens and to improve the growth of plants by inoculation of seed or soil with selected strains of micro-organisms; mainly bacteria. More recently, selected bacteria that are expected to degrade xenobiotics have been suggested for the bioremediation of polluted sites (Short et al 1990). Nowadays, risk assessment studies also need to address the fate of engineered telluric soil and non-telluric soil bacteria (Tiedje et al., 1989; Doyle etal, 1991).

Besides increased crop yields claimed by Russian workers (Schroth and Becker, 1990), most of the tentative applications of micro-organisms in agricultural soils have failed. The widely practised inoculation of legume seeds with Rhizobium spp. is one of the few examples of success of application of micro-organisms to improve crop yield (Stacey and Upchurch, 1984). Application of the strain K84, and more recently of the modified strain K1026, of Agrobacterium radiobacter to control crown gall of plants is one of the few examples of a biological control method commercially applied with some success in several countries (Ryder and Jones, 1990). In most cases, the beneficial effects expected from the microbial inoculation are not consistently reproduced under field conditions. The poor survival of the introduced microorganisms in soil is the main explanation for these failures. In fact, the population density of the introduced micro-organisms decreases to the limit of the carrying capacity of the soil. Moreover, the soil represents very heterogeneous environments in which the introduced micro-organisms must find suitable habitats, some of which are very strain specific (Hattori and Hattori, 1976).

Introduction

The spore-forming, Gram-positive bacterium. Bacillus thuringiensis (Bt), is a ubiquitous soil micro-organism with a world-wide distribution (Martin and Travers, 1989). Many thousands of isolates have been discovered since Ishiwata first isolated Bt from diseased silkworm larvae in 1901 (Nakamura and Dulmage, 1988; Beegle and Yamamoto, 1992; Feitelson et al, 1992), and it is possible to group these isolates into at least 34 serovars (also called subspecies) (de Barjac and Fraction, 1990). However, biomolecular techniques, such as multiplex polymerase chain reaction (PCR), permit a more rapid identification of strains (Bourque et at., 1993), and it is now possible to target specific DNA sequences with the use of this technology.

The delta-endotoxin produced by Bt is the most widely used biological pesticide – 2.3 million kg was used annually according to Rowe and Margaritis (1987) – and it is the ‘front-runner’ in attempts to commercialize biological control agents (BCAs); albeit that Bt is only a BCA in a limited sense, since it is the product of a living organism (the delta-endotoxin) which is utilized, rather than the organism itself.

The precise ecological role of this cosmopolitan invertebrate pathogen in the natural environment remains speculative (Smith and Couche, 1991). Bt reportedly does not produce spores or crystals in infected host cadavers, it is relatively poor at spreading natural infections in the field and rarely causes epizootics (Fuxa, 1989). Its ubiquitous occurrence in the soil (Martin and Travers, 1989), phylloplane microflora (Smith and Couche, 1991), and in man-made ecosystems (Meadows et al., 1992) suggests that it may have a primary function other than as a pathogen.

Introduction

The fungus Gliocladium virens Miller, Giddens and Foster is an important biological control agent (Papavizas, 1985). A formulation of this fungus (strain Gl-21) was recently registered with the US Environmental Protection Agency (EPA) by W. R. Grace & Co. Conn. The formulation was developed in cooperation with the Biocontrol of Plant Diseases Laboratory (BPDL), US Department of Agriculture (USDA) (Lumsden et al, 1991). It is intended for use against dampingoff diseases of vegetable and ornamental seedlings caused by the soil-borne plant pathogens, Pythium ultimum and Rhizoctonia solani in glasshouse operations (Lumsden and Locke, 1989). This fungus is one of the first to be registered for biocontrol of plant diseases and will soon be available in the US for commercial use in glasshouse applications under the trade name, GliogardTM

Certain criteria were considered important in the early stages of development of biocontrol agents (Lumsden and Lewis, 1989). In the development of a screening method for the selection of an appropriate micro-organism, the following points were considered. The screening would involve: (1) the use of a relatively uniform, commercially available soil-less medium that is used extensively in commercial glasshouses where the disease problem occurs; (2) targeted pathogens were selected that are important in the confines of a glasshouse where use of a biological control agent would probably be most successful because of a relatively uniform environment; (3) microorganisms indigenous to the US were selected because non-indigenous micro-organisms might be conceived as more likely problems for the US environment; (4) a single isolate of a biocontrol agent for control of both pathogens was preferred over a mixture of isolates; and (5) a high value crop, important in the ornamental production industry, was selected to defray the cost of development and registration.

Introduction

The number of plant protection products and active ingredients has been considerably reduced in Germany during the 5 years since the new Plant Protection Act (Gesetz zum Schutz der Kulturpflanzen) of September 1986 came into force. The number of registered plant protection products now amounts to 851 formulations with 216 active ingredients (18 August 1993, Fig. 28.1). The decline is a result of the consistent application of more stringent requirements for product registration.

In the coming years, the number of authorizations is expected to decrease further. Authorizations made before the Plant Protection Act of 1986 have expired and today's requirements for re-registration are tougher. Only about 25% of the plant protection products that were registered in West Germany in 1986, have already become subject to the new Plant Protection Act. In the new Bundeslander (Federal States) in eastern Germany, old registrations are valid until the end of 1995 with the exception of some products banned in Germany and some whose use is not acceptable. It is assumed that about 90% of them do not comply with the requirements for reregistration. Although the range of plant protection products has decreased in Germany, there was no marked effect on their usage until 1989. In the late 1980s consumption of active ingredients in West Germany amounted to about 36000 tonnes p.a., but dropped after 1989. About 33000 tonnes were sprayed in West Germany in 1990. In 1992 33 570 tonnes were sold in united East and West Germany, although the acreage nearly doubled after unification (Table 28.1).

Introduction

Direct observations of microbial growth in a large number of natural ecosystems have shown that the predominant populations are attached to surfaces where they grow in glycocalyx enclosed microcolonies that develop into adherent biofilms (Costerton et al. 1987; Cheng et al. 1991; Lappin-Scott et al. 1992). In various digestive tracts, this adhesion takes the form of very specific associations with insoluble nutrients (Cheng et al. 1977; Akin 1979; Cheng et al. 1991) to form particle associated microbial populations, and with tissue surfaces to form tissue associated populations (Cheng & Costerton 1980; Cheng et al. 1981a, b). A variation of this latter mode of growth is the association of a major microbial population with the mucous blanket of secretory intestinal tissue (Rozee et al. 1982). Bacteria of the ruminant digestive tract can be divided into three distinct subpopulations, (i) those associated with the digesta, (ii) those associated with gastrointestinal tissue and (iii) those associated with gastrointestinal fluid (Cheng & Costerton 1980; Cheng et al. 1981a; Czerkawski & Cheng 1988).

General principles of microbial ecology dictate that any organism will establish itself in a favourable nutrient niche and proliferate within a physiologically integrated community. Our observations of the growth of bacteria associated with feed particles indicate that each species adheres to its own particular insoluble substrate (e.g. cellulose, protein, starch) and produces enzymes to degrade insoluble substrates to soluble nutrients (Cheng et al. 1984; McAllister et al. 1990b).

Introduction

The use of purified water in various industrial and medical applications has increased dramatically over the past 20 years. These different applications often require varying levels of water quality. Each industry sets specifications for the acceptance of purified water quality based upon their product or process demands. The aggressive nature of ionically ultrapure water (18.2 Mohm cm) can contraindicate its use in, for example, stainless steel distribution systems which are often employed in the pharmaceutical industry. While the presence of trace levels of silica in condensate polishing loop waters creates great concern in the power industry, low level silica contamination of purified waters used in the production of medical devices or photomasks, for example, may not constitute cause for alarm. If any one group of contaminants can be viewed as ‘universal’ in their distribution, significance and recalcitrance, it is the bacteria and their by-products. Their role as purified water contaminants appears to cross all boundaries of purified water application and usage.

In the semiconductor industry, the demand for contaminant free water has, to a great extent, driven ultrapure water technologies. Indeed, water purification technologies have advanced to the point whereby levels of ionic, organic, and particulate contaminants can be reduced to concentrations below current analytical detection limits. Unfortunately, this industry's ability to detect and remove biological particulates and organics has not kept pace with the needs of today's sophisticated devices. Yang & Tolliver (1989) have noted that, at the 1 megabit level where minimum circuit feature size is typically 1 μm, device yields are limited by 0.1 μm jam and larger particles.

Introduction

The disposal of sulphide bearing mine wastes (tailings) as open deposits results in the formation of unique acid generating ecosystems (Mills 1985). Acid formation occurs via biooxidation of mineral sulphides which generates dilute solutions of sulphuric acid containing soluble metalions. The microorganisms (predominantly acidophilic thiobacilli) responsible for this mineral oxidation exist as discrete biofilms obtaining energy for their metabolism from the sulphide mineral surfaces which they colonize (Norman & Snyman 1987; Southam & Beveridge 1992). Most references to biofilms conjure up images of thick layers of bacteria (>100 μm) encased in capsular material. In this chapter we will employ the term ‘discrete biofilm’ to refer to the colonization of mineral surfaces by Thiobacillus ferrooxidans which typically exist as a single cell layer that is devoid of capsule. In this context a biofilm is simply a metabolically active surface at a solid solution interface.

Bacterial leaching produces acid mine drainage (AMD) which seeps into the surrounding aquatic and terrestrial environments. AMD is of environmental concern not only because of its acidity but also because of the soluble heavy metals which may enter the eukaryotic food chain (Schofield 1976; Tamm 1976; Koryak et al. 1979; Cloutier et al. 1986). Sometimes bacterial leaching can be exploited for industrial purposes, for example in the recovery of copper or uranium (Brierly 1982). Bacterial oxidation is, then, both an economic liability due to the legislated costs associated with AMD neutralization and an economic benefit as evidenced in industrial leaching systems. Clearly, an understanding of the fundamental aspects of bacteria mineral interaction is essential for the prevention of this deleterious environmental impact and for the promotion of these industrial bio oxidative processes.