European Commission

Harnessing the potential of genetically modified microorganisms and plants. Cover illustration: An arbuscule formed by an arbuscular mycorrhizal fungas as seen at scanning electron microscope. European Community

Harnessing the potential of genetically modified microorganisms and plants. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server (http://europa.eu.int).

Cataloguing data can be found at the end of this publication.

Luxembourg: Office for Official Publications of the European Communities, 1999

ISBN 92-894-0295-4

Reproduction is authorised provided the source is acknowledged.

Printed in Belgium Table of Contents

Introduction and Background 4 Biotechnology in Agriculture 4 Guidelines and Regulations for the Use of GMOs 5 The IMPACT Programme 5 Genetically Modified Biological Control Agents 9 The Development of Genetically Modified Biocontrol Strains With Increased Biological Control Efficacy 10 The Ecological Impact of Genetically Modified Biocontrol Strains 12 The Impact of Biological Control Agents on Arbuscular Mycorrhizal Fungi 16 Persistence of Released Biological Control Agents in Soil 17 Field Release of Genetically Modified Pseudomonas fluorescens F113 Strain 19 Biofertilizers and Phytostimulators 23 Azospirillum 23 The Development of GM Azospirillum Strains With Altered Phytohormone Production 26 Genetically Modified Rhizobium Strains With Increased Competitiveness 27 The Impact of GM Rhizobium Strains on Arbuscular Mycorrhizal Fungi 29 Field Release of Genetically Modified Rhizobium 31 Transgenic Plants With Novel Properties For Disease and Pest Control 33 Effect of Transgenic Plants on the Microbial Population 35 The Impact of Transgenic Plants on AM Fungi 35 Field Trial Evaluation With Transgenic Plants Expressing AFP Genes 35 Genetic Technologies for Fingerprinting 37 The Use of ARDRA and RAPD Within IMPACT 37 The Use of RAPD Analysis for the Determination of Pseudomonad Biodiversity on the Rhizoplane and in the Rhizosphere of Sugarbeet Inoculated With P. fluorescens F113 40 Molecular Based Techniques for Monitoring AM Fungi 40 Further Applications of ARDRA and RAPD 41 Executive Summary 43 Conclusions 45 Achievements of the IMPACT Programme and Future Directions 47 ANNEX I 48 The IMPACT Consortium 48 Introduction and Background

Modern biotechnology can be defined as any process whereby new strains of organisms with novel applications are produced through the manipulation of the genetic material (DNA) using innovative genetic engineering techniques. According to the European Commission "biotechnolo• gy has emerged as one of the most promising and crucial technologies for sustainable develop• ment in the next century". In 1995 in Europe, approximately 400,000 jobs were associated with biotechnology and the estimated value of products and services using biotechnology was esti• mated to be in the region of Euro 40 billion. By the year 2005 it is expected that, given improve• ments in the business environment, biotechnology activity will be valued at approximately Euro 250 billion and will have created more than 3 million jobs. The European Commission believes that biotechnology has the potential to create new products and processes, increase productivi• ty in existing industries, stimulate demand for highly skilled labour and raise the quality of life through improved health and reduced pollution.

The principal areas in which biotechnology will play a vital role include the pharmaceutical sector, the food and drinks industry, chemicals industry, and agriculture. Biotechnology is currently an integral part of the pharmaceutical sector with biologically produced chemicals accounting for 13% of all new chemical entities and 5% of world-wide pharmaceutical sales. Within the food and drink industry, biotechnology is largely confined to improving the performance of production processes such as the use of microbially produced enzymes. Expansion of biotechnology in the food and drink sector to include genetically modified crops for example, will largely depend on consumer acceptance and the demonstration of tangible benefits in products derived from biotechnology. The advantages that biotechnology offer the chemical industry has an added ben• efit to the environment. Novel genetically modified microorganisms have been developed for use in the manufacture of detergents, textiles and paper. Compared to naturally occurring microor• ganisms previously used by such industries, the genetically modified microorganisms do not pro• duce toxic or unwanted by-products and due to their increased efficiency, the demand for water, steam and electricity can be reduced by up to 50%. It is in the agriculture and food sector, how• ever, that major developments and growth are predicted. This brochure highlights progress that has been achieved in the agri-food sector with genetically modified microorganisms and plants within the framework of the European Union supported IMPACT1 project.

Biotechnology in Agriculture

Biotechnology has begun to revolutionize agriculture with strategies to develop new crops with higher yields, improved nutritional content and the ability to grow in unfavourable environments with reduced need for fertilizers and pesticides.

Genetically modified organisms (GMOs) are defined as organisms in which the genetic material has been altered or modified so as to create novel genetic combinations and properties. Essentially, modern scientific techniques allow the introduction of desirable traits into organisms by means of the specific transfer of genetic material (DNA). This new technology is leading to the development of novel crops, genetically modified for characteristics including resistance to pests and pathogens and tolerance of herbicides, and new microbial strains which, when added to the roots of plants, can protect the plant from disease and can increase plant growth. Given the

1. Interactions between Microbial inoculants and resident Populations in the rhizosphere of Agronomically important Crops in Typical soils. restrictions on chemical pesticides and fertilizers and the environmental implications of their use, biotechnology is providing new products for society for which, at present, there are no alterna• tives. Future developments within biotechnology are likely to lead to the development of new prod• ucts, which include crops with improved flavour and nutritional content and increased tolerance to drought and high salt concentrations allowing crops to be grown in a wide range of habitats. In addition, new microbial strains which can control disease and stimulate plant growth are expected to lead to a reduction in the use of pesticides, fungicides and fertilizers and provide new options for the control of crop diseases which currently cannot be managed even with exist• ing agricultural chemicals. In this way it is envisaged that biotechnology can contribute to the major European goal of environmentally friendly and sustainable food production.

Guidelines and Regulations for the Use of CMOs: As with all new technologies, it is natural that a number of questions concerning the development and use of genetically modified organisms in agriculture are addressed. The issues centre large• ly on the concerns regarding the long term effects that GMOs may pose to human health and the environment. Given these issues, many countries have introduced Biosafety Milex legislations. Within the European Union, EU Directive 90/220/EEC regulates the deliberate release into the environment of genetically modified organisms. Essentially this Directive ensures that GMOs be "subjected to satisfactory field testing at the research and developmental stage" and that prior to field release GMOs must be subjected to stringent laboratory testing to characterize the GMO (e.g. rate and level of expression of new genetic material, stability of the organisms in terms of genetic traits etc.) and to determine the behaviour and characteristics of the GMOs and their eco• logical impact as observed in simulated environments such as microcosms, growth rooms and greenhouses. A technical dossier relating to the GMO including foreseeable risks which the GMO may pose to human health or the environment is submitted to the Competent Authority of the Member State within whose territory the release is to take place (Table 1).

The Competent Authority has 90 days to accept or reject the application based on the compliance of the dossier with EU Directive 90/220/EEC and following an evaluation of the risks posed by the release. The Directive is currently under amendment to include even more stringent informa• tion requirements.

The IMPACT Project

The IMPACT project is an example of one of the EU research and development initiatives under• taken to facilitate the development of a scientific baseline to help with the biosafety assessment of GMOs destined for release into the environment. As previously mentioned, the principal con• cern regarding the release of genetically modified microorganisms and transgenic plants into the environment is their possible unforeseen effects on the native fauna and flora. For example, the planting of fields with transgenic plants resistant to insect pests may have an effect on popula• tions of non-target insects. Similarly, the introduction of microbial inoculants into soil for the con• trol of plant pathogens may also have unforseen effects on other groups of microorganisms including those which are actually beneficial to the plant. It is therefore essential to assess the impact of GMOs on native populations of plants, animals and microorganisms in the environment in which they are released.

The IMPACT project deals with the Interactions between Microbial inoculants and resident Populations in the rhizosphere of Agronomically important Crops in Typical soils. The rhizosphere is defined as the area of soil in direct contact with the plant root and is a site of intense micro- EU DIRECTIVE 90/220/EEC FOR THE DELIBERATE RELEASE OF GMOs INTO THE ENVIRONMENT

INFORMATION REQUIRED FOR NOTIFICATION

A: Characteristics of: i) The Donor ii) The Recipient INFORMATION RELATING TO iii) The Parental Organism THE GMO B: Characteristics of the Vector

C: Characteristics of the Modified Organism

INFORMATION RELATING TO THE A: Information on the release CONDITIONS OF RELEASE AND THE RECEIVING ENVIRONMENT B: Information on the environment (both the site and the wider environment)

A: Characteristics affecting survival, INFORMATION RELATING TO multiplication and dissemination TO THE INTERACTIONS BETWEEN THE GMOs AND THE B: Interaction with the environment ENVIRONMENT C: Potential environmental impact

A: Monitoring techniques INFORMATION ON MONITORING, CONTROL, WASTE TREATMENT B: Control of release AND EMERGENCY RESPONSE PLANS C: Waste treatment

D: Emergency response plans

Table 1: EU directive 90/220 for the deliberate release of GMOs in the environment. bial activity. The overall objective of the IMPACT project is to assess the effect that introduced microorganisms (wild-type or genetically modified) have on the resident microbes (bacteria and fungi) present in the soil associated with the roots of crop plants (including transgenic plants). The general research priorities set are the study of biological phenomena related to the release of GM microorganisms and novel plants into the environment and to investigate the ecological and environmental consequences on the resident microbiota in the rhizosphere.

IMPACT is a multidisciplinary project involving seventeen research and industrial centres of excel• lence in eight European countries. The expertise of the different centres involved in IMPACT cov• ers microbial ecology, molecular genetics, crop protection and agronomy and provides a compre• hensive approach to focus on relevant scientific issues ranging from genes to field testing GMOs {Figure 1 and Annex I). The IMPACT consortium allows close collaboration between the research institutions allowing large scale experiments and field trials to be conducted which, without col• laboration, would not be feasible. In addition, given the geographical spread of the research insti• tutions within the IMPACT programme, microbial inoculants can be tested in a range of soil types and climatic conditions. Such studies provide essential information on how modified and non- modified microbial inoculants behave under agronomic conditions in the European context.

The IMPACT project addresses key areas of agricultural and environmental biotechnology relating to GMO construction and use including the following key activities:-

7. Genetically Modified Biological Control Agents: Biological control agents may be defined as microorganisms (bacteria or fungi) that can antago• nize the agents responsible for plant disease (bacterial and fungal pathogens). The use of bio• logical control agents is seen as an environmentally friendly alternative to the use of chemical fungicides and pesticides. There are a number of ways in which biological control agents can con• trol plant pathogens including the production of anti-microbial compounds. Within the IMPACT con• sortium, GM biological control agents have been produced which have been modified for enhanced production of anti-microbial compounds. This modification increases the biological con• trol efficacy of these strains. However, it is recognised that in addition to the target pathogen, biological control agents may also affect the resident microflora including microorganisms that are actually beneficial to the plant. For this reason, the fate of GM biocontrol strains and their impact on the indigenous microbial population is a major objective of the IMPACT consortium.

2. Biofertilizers and Phytostimulators: Biofertilizers and phytostimulators are microorganisms that can directly promote plant growth. Biofertilizers include the bacteria of the genus Rhizobium, Sinorhizobium and Bradyrhizobium, which form symbiotic relationships with leguminous plants and provide nitrogen in a usable form to their host. Genetically modified biofertilizers have been developed, within the IMPACT pro• gramme, which are more effective at establishing symbiotic relationships with plants and so reduce the need to apply nitrogen fertilizers to the soil. The phytostimulators include Azospirillum bacteria, which produce plant growth stimulating factors. The plant growth stimulating factors increase plant root growth and allow increased uptake of water and nitrogen. Genetically modi• fied strains have been developed with enhanced plant growth factor production with the view to increasing crop yield and reducing pollution from fertilizer applications. Within IMPACT, in addition to the determination of the efficacy of GM biofertilizers and phytostimulators, their effect on the indigenous microbial population is being determined.

3. Transgenic Plants With Novel Properties For Disease and Pest Control: In order to reduce the need for the application of chemical fungicides, transgenic plants have been developed which produce biological anti-fungal proteins. The ability of these plants to con­ trol disease is being assessed within the IMPACT consortium. In addition, since the anti-fungal proteins are likely to come in contact with non-target microorganisms, their impact on the indige­ nous microflora is also being determined.

4. Genetic Technologies for Fingerprinting Microbial Communities in the Rhizosphere: One of the principal difficulties in the field of microbial ecology is the accurate identification and enumeration of key microbial species and the microbial population as a whole. Within the IMPACT consortium, the effect of genetically modified microorganisms on the native microflora is largely being assessed by a combination of classical microbiological and molecular techniques. Specifically, genetic fingerprinting is being applied for the detection and monitoring of genetically modified microorganisms and as a tool to assess the impact of introduced organisms (modified and non-modified) on native microbial populations.

IMPACT CONSORTIUM

RESEARCH ACTIVITIES

AZOSPIRILLUM STRAINS RHIZOBIUM STRAINS -ENHANCED 1AA -ENHANCED NODULATION PRODUCTION COMPETITIVENESS

TRICHODERMA STRAINS \ -ENHANCED CHITINASE GENETICALLY MODIFIED AFP AND HYDROLYTIC PHYTOSTIMULATORS ENZYME PRODUCTION \

GENETICALLY MODIFIED BIOCONTROL AGENTS \

COLONIZATION AND y PERSISTENCE IN SOIL PSEUDOMONAS STRAINS -ENHANCED ANTIFUNGAL METABOLITE PRODUCTION

IMPACT ON INDIGENOUS MICROBIAL POPULATIONS y / ν \

Figure 1: Research activities within the IMPACT consortium. Genetically Modified Biological Control Agents:

Biological control is generally defined as "any condition under which, or practice whereby, survival or activity of a pathogen is reduced through the agency of any other living organism (except man) with the result that there is a reduction in the incidence of the disease caused by the pathogen". This definition naturally implies that biological control as a means of increasing crop production is a long established practice and has its roots in traditional agricultural practices. The use of intercropping, flooding and rotations as well as the application of manure are all forms of clas• sical biological control which have their origins in pre-history. Such traditional agricultural prac• tices are examples of indirect biological control whereby the change in soil conditions leads to an alteration in the microorganisms present within the soil, the required result being the eradi• cation or control of the plant pathogen.

A well documented phenomenon in soil biology is the "suppressive soil", which may be defined as a soil in which a pathogen is present but is unable to cause disease despite favourable physical conditions. The reason for this is the presence of antagonistic bacteria and fungi which can either directly (e.g. parasitism, anti-microbial metabolite production) or indirectly (e.g. competition for nutrients) prevent the growth and/or activity of the plant pathogen. The identification of suppres• sive soils has led to the isolation of effective biological control agents. Unlike the traditional indi• rect forms of biological control, the isolation, characterization and subsequent use of bacteria I/fun• gal antagonists (i.e. biocontrol agents applied to the seed coat or as a foliar spray) offers a more specific form of disease control. Furthermore, subsequent investigation has led to an under• standing of the mechanisms by which such biological control agents prevent plant disease.

The biological control agent Pseudomonas fluorescens CHAO on the roots of tobacco.

Renewed environmental and public health concerns related to the widespread use of chemically synthesized pesticides has resulted in protective legislation governing the use of many pesticides. Among these is methyl bromide which is used as a broad spectrum pesticide, targeting many pests ranging from microbial pathogens, nematodes and insects. Such a pesticide has obviously enjoyed widespread use but, since its identification as a ozone depleting substance, it will be completely phased out by 2010. With additional more stringent controls expected in the near future, there is obviously the need to develop new and safer alternatives to chemical pesticides. Among the alternatives to chemical pesticides is the use of naturally occurring biological control agents, which represent an effective and environmentally acceptable strategy for the replace• ment, or decrease, of chemical pesticides. In addition, the knowledge obtained regarding the mechanisms whereby biocontrol ability is conferred, coupled with the recent advances in biotech• nology, has allowed the development of new genetically modified biological control agents which are more effective at controlling plant disease.

The Development of Genetically Modified Biocontrol Strains With Increased Biological Control Efficacy: Among the means by which biological control agents antagonize plant pathogens is by the pro• duction of anti-microbial compounds. Within IMPACT, a soil bacterium was isolated from the root hairs of sugarbeet. This bacterium, called Pseudomonas fluorescens F113, proved to be effec• tive at preventing the growth of a number of fungal pathogens under laboratory conditions. P. flu• orescens F113 produces a number of compounds which could have been responsible for the observed anti-fungal activity. Among these compounds is the anti-fungal metabolite 2,4- diacetylphloroglucinol (Phi). When a strain of P. fluorescens F113 was developed which was unable to produce Phi, the ability to prevent fungal growth was lost. Further evidence that Phi pro• duction is responsible for the observed anti-fungal activity was achieved by inserting the genes responsible for Phi production into a non-Phi producing bacterium. This strain which previously did not antagonize fungi, produced Phi and was effective at preventing fungal growth (Figure 2)

Conferring Antifungal Activity on the Phlorogiucinol Negative Strain, Pseudomonas fluorescens Ml 14, by Insertion of Phlorogiucinol Genes FromP. fluorescens Fl 13

Phlorogiucinol genes from I'. fluorescens F113 inserted into DNA pCUMJ carrier (Plasmid Vector pCU203)

Plasmid inserted into P. fluorescens Ml 14

Ml 14:Does not produce Phi. Cannol inhibit the growth of Pythium ultimum.

Ml 14 with plamid pCU203 can produce Phi and can inhibit Pythium ultimum

Figure 2: Conferring antifungal activity to a phlorogiucinol negative pseudomonad.

10 Within IMPACT, genetically modified Phi overproducing strains were developed to determine whether this would result In increased biological control efficacy (i.e. does Increased antifungal metabolite production result In increased plant protection from disease). The strategy for increas­ ing Phi production involved increasing the copy number of the Phi biosynthetic genes using a gene expression vector {Figure 3).

The Development of Genetically ModiftedPseudomonas fluorescens Fl 13 Strain for Overproduction of the Antifungal Metabolite Phlorogiucinol (Phi)

Biological control strain ( Pseudomonas fluorescensFl 13) Antifungal metabolite production

Gene carrier (plasmid)

Increased antifungal metabolite production 1Λ·^-

Figure 3: Development of genetically modified Pseudomonas strain.

A trial was conducted to determine whether the genetically modified strains were more effective biological control agents and whether they had an impact on native indigenous microorganisms. This trial involved the coating of sugarbeet seeds with the biological control agents (i.e. P. fluo­ rescens F113 and genetically modified strain F113:pCU8.3 which overproduces Phi) and growing them in soil naturally infested with the plant pathogenic fungus Pythium ultimum. The fungus Pythium ultimum is a common and destructive soil-borne plant pathogen which causes the dis­ ease 'damping-off on many agronomically important crops. The symptoms of the disease include the collapse of the lower stem of seedlings and rotting of seedling roots.

11 The inoculation of sugarbeet with the genetically modified strain resulted in increased protection from damping-off disease (Figure 4). The results of the microcosm trial demonstrated that new and Improved biological control agents can be produced through the development of genetically modified bacteria which show increased production of the anti-fungal metabolite Phi compared to the wild-type Phi producing strain, F113. An important observation is that the Phi overproducing strain conferred a level of biocontrol that was not significantly different from the fungicide treat• ed control. This implies that the coating of seeds with P. fluorescens F113 genetically modified for overproduction of Phi (i.e. strain F113:pCU8.3) is as effective at controlling damping-off dis• ease compared to the chemical fungicide. Therefore, not only can new and improved biological control agents be produced by genetic modification for increased anti-fungal metabolite produc• tion, but such strains can effectively compete with fungicides and could form the basis for new and environmentally friendly plant disease control products.

uu

95 S bc 90

75

70 Untreated Fungicide pi 13 Fl 13: Control Control pCU8.3

Figure 4: Effect of inoculation of sugarbeet seeds with Pseudomonas fluorescens F113 srains on plant health in Pythium ultimum infested soil. F113 is the wild type strain, producing normal amounts of 2,4- diacetylphloroglucinol; F113:pCU8.3 is the GM strain with enhanced Phi production. Values not followed by the same letter differ significantly at P-0.05.

The Ecological Impact of Genetically Modified Biocontrol Strains: Before proposing genetically modified biological control agents for large-scale use in agriculture, it is necessary to ensure that the new products do not have any adverse effects on the environ• ment. In agriculture, it is not unusual to apply the same chemical pesticide several times during the year and/or in successive years. Therefore, when dealing with the potential ecological impact of biological control agents, it is important to consider that the biocontrol agent may be applied several times to the same soil.

Within IMPACT, experiments were carried out to investigate the ecological impact of the biocon• trol agent Pseudomonas fluorescens CHA0-Rif(pME3424). This strain was obtained by genetic modification of P. fluorescens CHAO-Rif, a bacterium which occurs naturally in a Swiss soil. The

12 modified strain overproduces the two anti-fungal metabolites 2.4-diacetylphloroglucinol (Phi) and pyoluteorin (Pit). In biocontrol experiments, the genetically modified strain was more effective at controlling damping-off of cucumber (caused by Pythium ultimum) than the wild-type strain.

Pseudomonas fluorescens CHAO on the hyphae of T. basteóla

The principal objective of the impact studies was to determine whether repeated inoculations of the genetically modified biological control agent had an ecological impact on the microorganisms naturally present in the soil.

Cucumber was grown several times in pots treated with Pseudomonas fluorescens CHAO-Rif and the genetically modified P. fluorescens CHA0-Rif(pME3424) prior to each sowing. At the end of each cycle of cucumber growth, samples were taken to study whether treatments had an impact on the microorganisms naturally present in the vicinity of the root (i.e. the rhizosphere).

Repeated inoculations (five in a row) with the genetically modified biocontrol agent had no appar• ent impact on bacteria naturally present in the rhizosphere (Figure 5). At the same time, repeat• ed growth of cucumber in the same soil resulted in significant modifications in the bacterial com• munity of the rhizosphere. For example, the number of indigenous spore-forming bacteria in the rhizosphere was ten times lower at the end of the fifth cycle of cucumber growth than at the end of the first cycle. Therefore with the techniques employed, the inoculation of GM strains to soil had no apparent impact on microbial populations as opposed to the simple act of growing a plant in soil, which proved to have a major impact on the soil microflora.

Another technique for monitoring the impact of genetically modified organisms is the measure• ment of soil enzyme activities. Enzymes may be defined as proteins, produced by all organisms,

13 8

υth BD O

Culturable resident Culturable resident spore-forming bacteria fluorescent pseudomonads

Figure 5: The effect Pseudomonas fluorescens CHAO-Rif and its genetically modified derative CHA0-Rif(pME3424) on the culturable resident spore-forming bacteria and fluorescent pseudomonad populations in the rhizosphere of cucumber 32 days after sowing. Untreated control ( M); CHAO-Rif ( U); CHA0-Rif(pME3424) ( m) and Fungicide control (Ridomil, U).

which act as biological catalysts. A multitude of enzymes function in the rhizosphere and the mea­ surement of some representative activities can give an indication of the impact of inocula upon the essential reactions which provide nutrients to the plant. Furthermore, certain enzyme activi­ ties can be related to specific populations In the soil; thus changes in the activity of such enzymes gives an indication of perturbations In microbial populations. For example, acid phos­ phatase is predominantly produced by plants and fungi whereas alkaline phosphatase is pre­ dominantly produced by bacteria. Both of these enzymes are extremely important in the release of phosphate, a vital plant and microbial nutrient, from the soil. NAGase is another important soil enzyme which is involved in the breakdown of chitin (the predominant fungal cell wall component), and its activity is related to active fungal biomass.

The advantage of this technique over classical microbiological (e.g. enumeration of bacteria and fungi on agar plates) and molecular techniques Is that they give an indication of the overall effects of the inoculant on the rhizosphere ecosystem. Methods developed during the impact pro­ gramme have allowed soil enzymes to be measured In a simple, rapid and sensitive manner, giv­ ing it an advantage over classical biochemical methods.

Analysis of soil samples from field trials and glasshouse experiments conducted within the IMPACT programme have shown that the impact of commercial fungicide treatments on soil enzyme activities are far greater than the impact of genetically modified biological control agents (Table 2). Such a result indicates that the use of microbial inoculants, wild-type or genetically modified, offers an environmentally friendly method for the control of plant diseases. The com­ bined approach of measuring both bacterial numbers by classical microbiological techniques and the determination of soil enzyme activities offers a more comprehensive view of the ways an inoculum can Impact on the environment. The transnational structure of the IMPACT consortium

14 has allowed such studies to be conducted and will facilitate future trials to determine the impact of GM inocula in sites throughout Europe.

Seed Treatments Enzyme Fl 13 GM Fl 13 Untreated Control Fungicide (Phi producer) (Phi overproducer)

Acid Phosphatase 3.16 3.09 3.02 3.23 Alkaline Phosphatase 1.14a 1.36 b 1.23 a 1.36 b Aryl Sulphatase 0.17 0.20 0.20 0.20 Phosphodiesterase 0.26 a 0.31 b 0.28 ab 0.29 ab N-acetylglucosaminidase 0.28 b 0.20 a 0.23 ab 0.23 ab ß-glucosidase 0.23 0.22 0.22 0.24 Acid ß-galactosidase 0.20 0.21 0.22 0.23

Table 2: The effect of fungicide and Pseudomonas fluorescens F113 strains on rhizosphere soil enzyme activities. Enzyme expressed as mg pNP released / hour / g dry weight soil. Values in a row not followed by the same letter differ significantly at P=0.05.

The production of Phi in the rhizosphere has been monitored using a reporter gene fused to the biosynthetic gene phIA (i.e. a lacZ fusion). Experiments with this system have demonstrated that

Bacterial colonies on agar plates. Each colony arises from a single bacterial cell, known as a colony forming unit (CFU). By diluting down soil samples and plated out on agar medium it is possible to estimate the number of culturable bacter• ial cells in soil.

15 the plant species on which the inoculant is colonising can play a major role in Phi production. For example, phIA expression was significantly higher on the roots of the moncotyledons, maize and wheat, compared to the expression on the dicotyledons, cucumber and bean, and plant age was shown to have a significant affect on expression. In addition, phIA expression was markedly enhanced in the presence of the important fungal phytopathogen Pythium ultimum. This strategy for assessing the activity of biocontrol inoculants, developed within the IMPACT consortium, not only provides relevant information on anti-fungal metabolite production, but also provides key information on how the utilization of biocontrol agents can be optimized.

The Impact of Biological Control Agents on Arbuscular Mycorrhizal Fungi: A mycorrhiza is a sustainable, non-pathogenic interaction between a fungus and a plant root. In the majority of mycorrhizal associations nutrient exchange occurs. For example, carbon, in the form of sugars, can pass from the host plant to the fungus. In return, the fungus can provide the plant with immobile mineral Ions such as phosphate, ammonia and even nitrate, in water stress conditions. The fungal mycelium can be very extensive, forming a large area of contact with the soil for efficient nutrient foraging. Without their fungal partner, many plants are unable to acquire sufficient phosphate from the soil. Other known functions of mycorrhizas include protection against soil-borne pathogens, protection from water stress and improvement of nodulation and nitrogen-fixation in legume crops.

From the different types of mycorrhiza recognised, the most common are arbuscular mycorrhizas (AM), which are associated with crop plants. They occur in approximately two-thirds of all land plants and are found in ecosystems ranging from the arctic to the tropics. The plant partners of these symbioses embrace many important crop species including cereals and many dicotyledo• nous crops such as legumes (beans, peas etc.) and potatoes. The fungal symbionts all belong to a small group of Zygomycete fungi of the order Glomales, which originated millions of years ago. The earliest fossil evidence of AM fungi comes from roots of Aglaophyton major, from the Rhynie Chert, dating from 410-360 million years ago. This is consistent with phylogenetic work based on ribosomal DNA genes, which estimates the origin of AM-like fungi at 462-353 million years ago. Both support the hypothesis that the evolution of AM symbiosis in early plants may have enabled plants to subsequently colonize the land.

Germinating spore of the AM fungus Gigaspora margarita.

16 Given the importance of AM fungi, it is essential to determine the effects of normal agricultural practice and GMOs on the symbiotic relationship between AM fungi and plants. For example, an important question is whether genetically modified biocontrol agents and transgenic plants pro• ducing anti-fungal metabolites can affect the initiation of the symbiotic relationship. A series of growth room and glasshouse experiments was therefore conducted within the IMPACT consortium to assess the impact of GMOs on mycorrhizal formation.

Biocontrol strain Pseudomonas fluorescens F113:pCU203 was assessed for its impact on the representative AM fungus Glomus mosseae. The genetically modified strain which overproduces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phi), had no effect on spore germination of G. mosseae, compared with the wild-type P. fluorescens F113 and the Phi negative strain P. fluroescens F113G22. In addition, the Phi overproducer did not affect mycelial growth compared with the untreated control (Table 3). This result suggests that despite the anti-fungal activity of Phi, the biological control strain does not adversely affect this natural and essential symbiotic relationship and clearly demonstrates that there is major potential for the exploitation of geneti• cally modified biocontrol agents as components of integrated pest management strategies being developed for more environmentally friendly food production.

% Spore Germination Mycelial development Pseudomonas strain In vitro In soil (mm/germinated spore)

None (control) 70 a 60 a 10 a

Fl 13 (Phi positive) 69 a 62 a 33 b

Fl 13G22 (Phi negative) 72 a 67 a 47 c

F113(pCU203; Phi over- 71 a 64a 15 a producer)

Table 3: Effects of Pseudomonas fluorescens strains on Glomus mosseae spore germination in vitro and in soil and on mycelial growth in vitro. Values in a column not followed by the same letter differ signifacantly.

Persistence of Released Biological Control Agents in Soil: The fate of bacterial inoculants in natural environments is a key issue addressed in internation• al regulations. In a long term field experiment it was determined that, after several months fol• lowing inoculation into soil, less than 0.1% of released biological control bacteria were detected using conventional monitoring methods (Table 4). Using a microscope based technique (immuno• fluorescence microscopy, Figure 6) in which bacterial cells can be more accurately determined, the number of cells was substantially higher. Furthermore, when this technique was combined with a test to determine the number of living bacterial cells, It was possible to determine the num• ber of released bacterial cells that were actually still alive. By this approach it has been shown that bacterial cells released into the environment can exist in three physiological states:

a) Bacteria that can be grown in the laboratory (i.e. culturable). b) Bacteria that are not culturable but are still alive, known as the Viable But Non- Culturable state (VBNC).

17 c) Bacteria that are neither culturable or viable (considered to be either dormant, inactive or dead)

The significantly higher level of VBNC cells compared to culturable cells has led to much research being conducted within the IMPACT programme to determine the ecological role of these non-cul- turable bacteria in the soil ecosystem and to understand the factors which induce the VBNC state. Among the factors which have been shown to induce VBNC are oxygen limitation, a decrease in soil pH (i.e. an increase in soil acidity) and exposure to high concentrations of salt (NaCI). Therefore, it would appear that certain soil microniches may favour the occurrence of non- culturable states.

Some genes responsible for the induction of VBNC state due to aforementioned factors have been identified and mutant strains defective in these genes have been developed. Ongoing experiments dealing with the VBNC state in GM and mutant strains will be of major importance when consider• ing the formulation of agricultural products containing biocontrol bacteria.

Status of Cells Yc Total Cell Number

Culturable Cells 0.08%

VBNC Cells 4.77%

Dormant / Non Viable Cells 95.2%

Table 4: Physiological status of Pseudomonas fluorescens CHAO-Rif cells 200 days after Inoculation.

Figure 6: The use of immunofluorescence microscopy for viewing bacterial cells.

18 Field Release of Genetically Modified Pseudomonas fluorescens Fl 13 Strain: In order to track and monitor bacterial strains in field conditions they need to be marked. For labo­ ratory experiments it is possible to insert antibiotic resistance genes into the bacterial strains. Only the bacteria resistant to the antibiotic will grow on growth media containing the antibiotic; thus one can accurately determine the number of antibiotic resistant cells (i.e. marked cells) on, for example, plant roots. For field experiments, however, it is unacceptable to release bacteria which are resis­ tant to clinically relevant antibiotics since there Is the possibility that such traits could be transferred to other bacteria and lead to the emergence of undesirable antibiotic resistance in nature.

Alternative tagging systems have therefore been developed. One of the most useful is the intro­ duction of the genes that allow the bacterial cell to catabolize the sugar lactose. Since certain bacterial groups, such as the pseudomonads, do not ordinarily have the ability to utilize lactose, Pseudomonas fluorescens F113 with inserted lactose catabolism genes (i.e. lacZY genes) can be easily monitored in the field (Figure 7)

Marking Pseudomonas fluorescens F113 with lacZY for monitoring GM strains in the field

Escherichia coli

The genes lacZ and lacY. required for the utilization of lactose, are removed from E. coli and cloned into Ρ. fluorescens

Pseudomonas fluorescens F113

Pseudomonas fluorescens F113 with lacZY genes When bacterial cells are plated onto agar medium containing the substrate X-gal, cells which can catabolize lactose form blue colonies

Figure 7: Marking Pseudomonas fluorescens F113 for monitoring purposes In the field.

19 Within the IMPACT project, a field trial was conducted to determine the impact of the genetically modified strain P. fluorescens F113/acZY on the natural soil microflora and to assess the fate of the GM strain in the field. This field trial to assess the release of a GM bacterial strain was one of the first conducted under the guidelines specified within the EU Directive 90/220 (field trial plan, Table 5).

Field trial plan for sorghum with GM Azospirillum brasilense inoculants and soybean with GM Pseudomonas fluorescens inoculants

Sorghum

B la 2a 3a 4a B B 4a 2a la 3a Π

B Ib 2b 3b 4b B B 4b 2b 1b 3b B

B le 2c 3c 4c B B 4c 2c le 3c B 8m ~J_ B 2a 4a la 3a B B 2a 3a 4a la B

B 2b 4b lb 3b B B 2b 3b 4b lb B B = Bonder Plants

B 2c 4c le 3c B B 2c 3c 4c le B

Ä Soybean •I Β 1 2 3 4 Β Β 1 3 4 2 Β

Β 3 2 4 Ι Β Β 2 1 3 4 Β

Sorghum: Factors under study: F.l) Inoculant type - 4 levels 1 - Control - no inoculant 2 - Azospirillum brasilense / Sp245 lacZ 3 - Azospirillum brasilense I Sp6 gusA

4 - Azospirillum brasilense I Sp6 (IAA++) gusA

F.2) Nitrogen Fertilization - 3 levels a - Control - no fertilizer b - 70% of optimal dose c -100% of optinal dose

Soybean: Factors under study: F.l) Inoculant type - 4 levels

1 - Control - no inoculant 2 - Bradyrhizobium japonicum (commercial strain) 3 - B. japonicum + Pseudomonas fluorescens F113 lacZY 4 - Pseudomonas fluorescens Fl 13 lacZY

Table 5: Field trial plans for the release of genetically modified inoculants developed within the IMPACT consortium.

20 Results from the trial showed that the genetically modified strain, Inoculated onto soybean, resulted in no observable negative effects on any crop yield parameter. These included seed yield, protein and oil content of the seed and biomass and nitrogen content of plant residues. A detailed analysis of the colonization of soybean roots by indigenous bacteria, showed that inoc­ ulation with P. fluorescens F113/acZY had no negative impact (Figure 8). Furthermore, the inocu­ lated strain significantly increased the nodulation of soybean by Bradyrhizobium japonicum (i.e. the inoculant did not interfere with the symbiotic relationship between the plant and the nitrogen fixing bacterium, Figure 9). The lacZY marker gene allowed accurate measurement of the inocu­ lant which, under field conditions, proved to be as effective at colonizing the roots of soybean as the wild-type strain P. fluorescens F113 (i.e. the genetic modification did not compromise its abil­ ity to grow on soybean roots).

The numerous microcosm, glasshouse and field trials conducted within IMPACT, employing the techniques for monitoring the release of GM microorganisms and their effect on the resident microflora, has provided strong evidence of both the effectiveness of these strains as biological control agents and their lack of measurable ecological impact on the microbial population com­ pared with-wild type strains. Furthermore, vital baseline information has been established with the field release of Pseudomonas fluorescens F113/acZY, allowing the accurate assessment of the ecological impact of new bacterial strains genetically modified for increased biological control efficacy.

9.0

8.0

7.0

6.0

5.0 © 4.0 f 3.0 υ 2.0 1.0

0.0 Total Aerobic Microfungi Streptomycetes Fluorescent Aerobic Spore Bacterial Pseudomonads Forming Bacteria Population

Figure 8: The impact of Pseudomonas fluorescens F113LacZY, inoculated with Bradyrhizobium japonicum, on the microbial population on soybean at harvest. Untreated control (M); P. fluorescens F113LacZY and B. japon­ icum (M). Error bars represent standard deviation.

21 6-1 b

5- a a a υ Ι­ Ο 4- (Ζυ) c 'S 3- ce 3 ■Ό Ο 2- Ζ

1-

Control Β. japonicum P. fluorescens B. japonicum + F1131acZY P. fluorescens FlUlacZY

figure 9: The effect of Pseudomonas fluorescens F113LacZY on the ability of Bradyrhizobium japonicum to nodulate soybean. Soybean inoculated singly with P. fluorescens F113LacZY and B. japonicum and co-inoculated with both strains. Values with same letter are not significantly different at P=0.05.

The genetically modified Phi overproducing strain P. fluorescens F113Rif pCUGP has been assessed in a field trial to determine whether the strategy employed for increasing anti-fungal metabolite production can affect maize yield in a soil where yield is often limited by a complex of minor soil-borne pathogens. The impact of this strain on the indigenous AM fungal population was also assessed. P. fluorescens F113Rif pCUGP, which contains multiple copies of the Phi biosyn- thetic locus, is marked with the gusA reporter gene which confers ß-glucuronidase activity on the inoculant. Strains containing gusA are blue in colour when grown on media containing the sub• strate X-glcA. Studies on the AM fungal population showed that neither P. fluorescens F113Rif pCUGP nor the wild-type control P. fluorescens F113Rif had an affect on (i) the average species richness, (ii) the total spore number, {\\\) the Shannon-Wierner diversity index, (iv) the percentage

Treatment Grain Yield (kg/plot)

Untreated Control 8.9 Fungicide Control 8.4 P. fluorescens Fl 13Rif (WT) 9.2 P. fluorescens F113Rif:pCUGP (GM) 9.7

Table 6: Effect of inoculation with Pseudomonas fluorescens biological control agents on maize yield

22 of maize roots that became mycorrhized and (v) the relative density of the AM fungal ecotypes. Furthermore, inoculation of maize seeds with P. fluorescens F113Rif pCUGP resulted in a signif• icant increase in maize yield compared with the untreated and fungicide treated control plants (Table 6). This result demonstrates the potential of GM microbial inoculants as alternatives to environmentally or health hazardous chemical fungicides and, as such, represent alternative strategies that will contribute significantly to sustainabillty within the agri-food sector.

Biofertilizers and Phytostimulators

Except for carbon dioxide (CO2) which plants obtain from the atmosphere, plants get all their nutri• ents from soil. Since the soil has to be amended with nutrients, it is highly desirable to supply these plant nutrients by means of renewable resources and in line with the current policy of sus• tainable agriculture.

In this respect, many lessons can be learned from nature. The best documented example of this principle is biological nitrogen fixation in leguminous plants. Certain bacteria can form nodules on the roots of plants such as beans, soybeans and clover, and supply the plants with nitrogen taken from the atmosphere. Although this symbiotic relationship is far from optimal, an important principle is illustrated: biological nitrogen fixation is a self-regulating system, whereby excessive nitrogen (in the form of ammonia or nitrate) leaking into the surface water is avoided. Nitrogen-fixing bacteria can be regarded as a self-propagating source of nitrogen for plants. Unfortunately, not all plants are able to initiate a successful interaction with nitrogen-fixing bac• teria. Grasses and cereal plants such as rice, wheat and maize do not directly benefit from the nitrogen-fixation capacity of the symbiotic soil bacteria, despite the fact that such bacteria are present in high numbers on the roots of these plants.

Until new technology and strategies are developed allowing symbiotic relationships between nitro• gen-fixing bacteria and these agronomically important crops, production yields will largely depend on input of chemical fertilizers. Nitrogen fertilizer is mainly supplied as nitrate which is very mobile in the soil. This explains why much more nitrogen is added to soil than is required for opti• mal plant growth. For example, for the growth of rice in paddy soil, it is current practice to add 450 kg N/ha while only 200-250 kg N/ha is taken up by the plant. Not only is half of the nitro• gen supplied lost (economic cost), but this also causes serious problems for the environment, through leakage in surface and ground water and accumulation of nitrogen oxides in the atmos• phere (environmental cost).

Different strategies to assure a better uptake of fertilizer by plant roots have been developed. These include other formulations of fertilizer (e.g. slow-release fertilizer) and the use of Plant Growth Promoting Rhizobacteria (PGPR). PGPR can exert their effect in an indirect way (e.g. biocontrol of pathogens and deleterious microorganisms, as discussed in the previous section) and in a direct way.

Azospirillum: The best documented example of PGPR acting in a direct plant growth promoting way is phy- tostimulation. Bacteria such as Azospirillum produce plant growth stimulating factors (auxins, cytokinins etc.) and when colonizing the roots of plants, can promote root growth (Figure 10). This assures a better uptake of water and nutrients by the plants and can result in higher crop yields. Furthermore, a better uptake of nitrogen results in less nitrogen remaining in the soil and con• sequently reduces the potential for ground water pollution (Figure 11).

23 Figure 10: Increased root growth as a result of inoculation with Azospirillum.

Figure 11: Reduced nitrogen content in soils containing plants with Azospirillum.

24 As with the biological control agent Pseudomonas fluorescens F113, genetically modified Azospirillum strains, marked with non­antibiotic resistance markers (I.e. genes which allow the bacteria to be monitored in the soil), were developed within IMPACT to assess their growth in soil and their effect on the grain yield of sorghum. In multiple field trials involving industry and research partners within the transnational framework of the IMPACT consortium, commercial scale trials were performed. The colonizing ability, persistence and survival of the genetically modified bacteria on the roots of sorghum and their effect on sorghum grain yield and the indige­ nous microflora was assessed. Furthermore, sorghum was sown in three plots amended with dif­ ferent levels of nitrogen to assess the effect of inoculants at different nitrogen concentrations.

The two strains under investigation were Azospirillum brasilense Sp6, producing normal levels of the plant growth stimulating factor IAA, and Azospirillum brasilense Sp6 IAA++, an IAA overpro­ ducing strain. The field release conducted within IMPACT was carried out according to the guide­ lines specified within the EU Directive 90/220 (field trial plan, Table 5).

Results indicate that Azospirillum inoculants significantly enhanced sorghum root development and increased grain yield (Figure 12). Such an observation demonstrates that the same grain yield can be obtained with reduced levels of nitrogen by inoculation of seeds with Azospirillum.

Persistence studies demonstrated that when seedling colonization by the genetically modified strain was satisfactory, cell density on the roots was at a maximum 15­20 days after sowing; from this point colonization of Azospirillum progressively decreased and, at harvest, cell density was very low (9xl03 cells/g soil dry weight). In addition, inoculation of genetically modified Azospirillum strains did not affect the indigenous microbial population (Figure 13).

9 a

8.5

8 ^b

7.5

7 Yiel d t/h a

6.5

Grai n 6

5.5

5 0 80 160

Nitrogen Added To Soil (N Kg/ha)

Figure 12: The effect of Azospirillum brasilense Sp6 (IAA producer) and A. brasilense Sp6 IAA++ (IAA overproducer) on sorghum grain yield. Untreated control (♦); Sp6 (β) and Sp6 IAA++ (»). Valuesnot followed by the same letter differ significantly at P=0.01.

25 9.0

8.0

7.0

oM 6.0 m rW" Η '^^ — 50 OC 4> Ê 4.0 U>-> Q 3.0 Wo3 ec ?0 NJ Ufa υ 1.0

0.0 Total Aerobic Microfungi Streptomycetes Fluorescent Aerobic Spore Bacterial Pseudomonads Forming Bacteria Population

Figure 13: The impact of GM Azospirillum brasilense strains on the microbial population 120 after sowing. Untreated control iß); A. brasilense Sp6 (M); A. brasilense Sp6 (IAA++, ■ Error bars represent standard deviation.

The Development of G/W Azospirillum Strains With Altered Phytohormone Production: Optimizing and exploring the principle whereby Azospirillum strains can promote plant root devel­ opment and increase nitrogen uptake, requires a better understanding of the mechanisms by which, and the conditions under which, these bacteria produce phytohormones. In addition, an understanding of the interaction itself between bacteria and plant roots is essential.

The three principal tasks within IMPACT in order to address these questions are:

a) To gain an understanding of the genetics and biochemistry of the synthesis of indole­3­acetic acid (IAA, the plant growth promoting hormone produced by Azospirillum).

b) To construct genetically modified Azospirillum stains with known production levels of IAA (i.e. IAA­minus, IAA­attenuated, IAA­over producers).

c) To test the effect of these genetically modified bacteria on plants (growth promotion, nitrogen uptake) and on the environment (interaction with resident microbial flora, survival and spread) under field conditions.

These worktasks have been translated into a research plan that involves bacterial engineering, physiological tests and field testing. The biosynthesis of IAA by Azospirillum turned out to be a complex process, mediated by at least three biosynthetic pathways. Altered IAA production (i.e. enhanced and reduced production) has been achieved by targeting the gene ipdC which codes for the key enzyme in the major biosynthetic pathway (the indole­3­pyruvic pathway).

26 In order to use these strains in field trials, they have been equipped with marker genes that allow the detection of the strains in the soil. The two marker genes being used are lue and gfp; the lue gene causes the bacterial cell to glow (bioluminescence) and the gfp gene (which codes for the green fluorescent protein) causes cell fluorescence.

Presently, Azospirillum strains with these basic features are available. However, before they can be considered for field release, extensive and careful testing under containment is required. Presently within IMPACT, research is focusing on the impact of genetically modified Azospirillum strains on indigenous microbial populations, plant growth and nitrogen uptake rates from soil. These studies are being conducted in microcosm experiments (i.e. growth cabinet and glasshouse studies) in order to gain vital information on the way GM strains are likely to behave under field conditions. The transnational partnership of the IMPACT consortium facilitates such experiments to be conducted with a range of crops, soil types and climatic conditions, repre• senting the breadth of agricultural parameters existing within Europe.

Genetically Modified Rhizobium Strains With Increased Competitiveness: The productivity of leguminous crops, which include beans, peas and clover, can be improved by inoculation of seeds with highly efficient nitrogen fixing bacteria. Legume inoculation, however, is often unsuccessful because of the occurrence of native soil bacteria with low nitrogen-fixing effi• ciency that can out-compete the introduced strains in terms of nodulation initiation. The ability to dominate nodulation is termed competitiveness and is critical for the successful use of rhizobial inoculants. Therefore, it is desirable that the inoculant strain be modified to ensure that it will occu• py a sufficient number of root nodules to provide high rates of nitrogen fixation for the plant host.

Within IMPACT it has been demonstrated that nodulation competitiveness of several Sinorhizobium meliloti strains from diverse geographical origins can be enhanced by genetic manipulation. This genetic manipulation consists of the modified expression of the nifA gene which is responsible for the control of all the nitrogen-fixation genes (nif genes). Mixed inocula• tion experiments in which wild-type and genetically modified S. meliloti strains compete for nodu• lation of the same host plant showed that the genetically modified strains occupy most of the

Rhizobium Nodule Occpancy Strains in Coinoculation (%)

2011 5 2011-GM 95 L5.30 22 L5.30-GM 78 GR013 7 GR013-GM 93 Rm41 13 Rm41-GM 87

Figure 14: Competitiveness of genetically modified (GM) Rhizobium strains compared with wild-type (WT) parental strains. GM and WT strains were co-inoculated In equal concentrations.

27 nodules on alfalfa roots (Figure 14). The genetic basis for this improvement is not well under• stood, but it has been hypothesized that nifA regulates the expression of genes other than nif genes and that modified expression of such genes will provide an advantage during nodule for• mation and development.

Other factors contributing to nodulation competitiveness include the ability of Rhizobium strains to effectively recognize the plant root. It has been hypothesized that the use of bacterial inocu• lants which are attracted specifically to target roots could allow more efficient inoculation, thus requiring lower doses of the bacterial strain. The role of bacterial movement towards roots on the competitive ability of strains has been analyzed using Rhizobium leguminosarum strains engi• neered to express ß-glucuronidase, a reporter gene which allows easy detection in nodules (the gene is known as gusA). By comparing the percentage of nodules induced by the wild-type, gusA- labeled strain compared to the nodules induced by a flagella-deficient non-motile strain (a flagel- lum is a whip-like structure which is responsible for propelling the bacterium through water), it has been shown that the presence of functional flagella is required for effective competition for nodulation (Figure 15).

Phenotype of a flagella-deficient mutant ofl?. leguminosarum

-non motile in liquid and solid culture

- S warming-deficient

-Nod Fix on peas

- LESS COMPETITIVE THAN WILD TYPE

Analysis of competitiveness of a flagella - deficient mutant

nodules W.T. gusA I mutant ratio % W.T. blue white

10:1 375 1 100 1:1 223 95 70 1:10 267 75 78

Values are the average of three replicates from indepepiaata

Figure 15: Decreased competitiveness of flagella-deficient Rhizobium leguminosarum mutants.

The first stage in the movement of Rhizobium towards the plant root, however, Is the recognition of molecules produced by the plant root. Mcp proteins (Methyl-accepting chemotactic proteins) are proteins in the cell walls of Rhizobium cells which sense molecules produced by the plant and initiate bacterial movement towards the plant root (Figure 16). Mcp-like genes have been identi• fied within R. leguminosarum and currently the plant-produced compound recognized by the Mcp

28 Figure 16: Movement of Rhizobium towards plant roots. Methyl accepting Chemotaxis proteins recognise specific root exudates (represented in the diagram by the same color).

protein is being identified. This will give the necessary information regarding the mechanism of root attraction allowing the development of Rhizobium strains with enhanced nodulation compet• itiveness and increased host specificity.

The Impact of CM Rhizobium Strains on Arbuscular Mycorrhizal Fungi: As discussed previously, the AM fungi are an important group of fungi that form symbiotic rela• tionships with plants. Within the IMPACT project it is important to determine whether Rhizobium strains modified for increased competitiveness resulting in increased colonization and nodulation of the plant root has an effect on the ability of mycorrhizal fungi to infect the plant root and thus establish the beneficial symbiotic relationship.

In a series of growth room and glasshouse trials it has been established that the GM Sinorhizobium meliloti strain, with improved nodulation ability, did not interfere with any aspect of mycorrhiza formation by the representative AM fungi Glomus mosseae. Indeed, the GM S. meliloti increased the number of AM colonization units and the nutrient acquisition ability of the mycorrhizal plant compared to the wild-type isolate (Figure 17 and 18). The establishment of the symbiotic interactions also induced changes in root morphology; in particular, the degree of branching and the number of lateral roots was higher in mycorrhizal plants inoculated with the genetically modified S. meliloti strain.

29 Figure 18:ComparativeeffectsofthetwoSinorhizobiummeliloti strains,thewildtype(M)anditsgeneticallymodified Figure 17:ComparativeeffectsofthetwoSinorhizobiummelilotistrains,wildtype(M)anditsgeneticallymodified differences betweentheeffectofGMandWTaresignificantat 5%level. Data aregivenonaperplantbasis.Thesymbol(*)denotesthat foreachparameterandharvesttime,the derivative (Ejonnitrogenandphosphorusaccumulation,inmycorrhizal (Glomusmosseae)alfafaplants. the effectofGMandWTaresignificantat5%level. a perplantbasis.Thesymbol(*)denotesthatforeachparameterandharvesttime,thedifferencesbetween derivative (a)on"entrypoint"formation,inmycorrhizal(Glomusmosseae)alfafaplants.Dataaregiven

Myconhizal Entry Points (No.)

— to u) è Sí Çb 5j 08888888 Time AfterInoculation(Weeks) Time AfterInoculation(Weeks) 2 46810 * 30 • • * Field Release of Genetically Modified Rhizobium: Genetically modified Rhizobium leguminosarum bv. viciae strains, marked with the lacZ gene and HgCb resistance genes (mer genes), were developed within the IMPACT consortium and their impact and fate under field conditions was assessed. In the field trial three GM derivatives of R. leguminosarum bv viciae 1003 (wild-type) were used; strain 1110 which contains plasmid pDG3 carrying genes for resistance to HgCb (mer genes) and lacZ whose expression is under the con• trol of the lacl-lacO system, strain 1111 carrying the plasmid pDG4 in which the lacZ gene is con- stitutively expressed at high levels and strain 1112 containing a copy of mer genes and a regu• lated lacZ gene inserted into the chromosome. GM strains were monitored according to the reporter system used (lacZ/mer) and populations were determined by the most probable number technique (MPN). Microbial activity in soil was assessed by monitoring CO2 which gives an indica• tion of soil metabolic activity and N2O emissions which is a measure of nitrogen transformation.

The presence of inoculant strains in the rhizosphere of pea plants was determined 10 days after sowing (Table 7). It was observed that all strains tested colonized the rhizosphere to the same extent. As for stability, revertants from strain 1110 and 1112 (both strains having regulated gene expression) were not detected by the plate count method, whereas a marked instability, due to the constitutive expression of lacZ, was observed with strain 1111.

CO2 emission in non-rhizosphere soil was shown to be significantly lower than in rhizosphere soil from un-inoculated plants. The latter had a lower respiration rate than the rhizosphere soil of inoc• ulated plants. However, CO2 production for soil inoculated with GM and non-modified strains was almost identical. These results indicate that although the presence of the plant had a consider• able impact on carbon mineralization in soil, the impact of GM Rhizobium strains is indistin• guishable from the impact of the wild-type strain. With regard to N2O emission, soil without plants

Rhizobium leguminosarum bv. viciae Strain Total Genetically Modified Revertants

1003 1.9x104

1110 3.4x105 3.4xl05 <102 1111 1.3x105 9.9x104 2.6x104 1112 2.9x104 2.9x104 <102

Table 7: Population density of modified and non-modified R. leguminosarum bv. viciae in the rhizosphere soil at emer• gence of pea plants (10 days after sowing). The initial inoculum was 6.03x10s (1003), 4.88x10s (1110), 2.27x10s (1111), 7.05x10s (1112) cfu/seed. Data are expressed as cfu/gr-' soil d.w. and represent the mean of three replicates for each treatment from two independent counts.

31 had a pattern of N2O production significantly different from soil with plants (Figure 19). N2O pro­ duction, however, was not significantly different between inoculated and uninoculated plants or between GM and non­modified strains. These results corroborate those on CO2 emission and sug­ gest that the impact of the plant on microbial activity is considerably greater than the impact of GM inoculants compared with wild­type strains.

270

260

^ 250 ce "β ■C. 240 'S ■Bulk Soil 03 230 -Not Inoculated ■Strain 1003 O 220 -Strain 1110 -Strain 1111 5 210 •Strain 1112 O =L 200

190

180

170 10 15 20 25 30 35 40 45 50 55 Time (Days)

Figure 19: N2O production, as a measure of nitrogen transformation, in soil planted with pea and inoculated with GM Rhizobium leguminosarum bv. viciae strains. Bulk soil: soil without pea. The initial inoculum was 6.03x10s (1003), 4.88x10s (1110), 2.27x10s (1111), 7.05x10s (1112) cfu/seed.

32 Transgenic Plants With Novel Properties For Disease and Pest Control

During its lifetime, a crop plant is under constant threat from a bewildering array of potential fun• gal pathogens. Disease is rare, though economically very significant, so the defence mechanisms evolved by the plant are generally effective against fungal colonization. For decades, plant breed• ers have used the Inherent resistance of plants to fungal pathogens to improve crop plants, often by introducing genes from related wild species. However in crops where little or no resistance to a pathogen is available or where resistance breaks down due to the appearance of virulent races of the pathogen, yield losses can be severe. The persistent losses In the European potato crop due to late blight caused by the pathogen Phytophthora infestans, and the recent epidemics of the same disease in the USA are examples where lack of resistance in the crop and increased virulence in the pathogen have combined to devastating effect.

In recent years, the revolution in DNA technology and genomic science, has provided the oppor• tunity to modify plants for a range of characteristics including resistance to pests and pathogens (Table 8). Within the last year, the majority of GM crops which have been granted market consent within Europe have been modified for resistance to herbicides (Table 9). This allows the farmer to apply a single broad spectrum herbicide to replace the use of several herbicides. In the fungal control area, the availability of a number of genes which encode potent anti-fungal proteins and continuing yield losses in crops that amount to almost 20% of production world-wide is a strong incentive to exploit the potential offered by the new genetic technologies. Whilst to date there are no varieties on the market with improved fungal resistance derived by transgenic modification, the evident successes of several companies with insect and weed control products, and the need to provide growers with alternative solutions to problem diseases, underlines that this will be an area of intense research and development Interest in the coming years.

GM plants or GM Developmental Company plant products Status

Herbicide Tolerant Plants In Commerce Monsanto,Calgene, Du Pont, Insect Resistant Plants In Commerce Monsanto, Mycogen Novartis Delayed Ripening In Commerce Agritope Inc, Calgene Virus Resistant Fruits and In Commerce Asgrow Seed Co Vegetables

Salt and Drought Field Trials Resistant Plants Fungal Pathogen Experimental Resistant

Nitrogen Fixing Cereals with Early Research Legume Genes

Edible Vaccines Research Secondary Metabolites, Research e.g. vitamins, enzymes.

Table 8: The developmental status of transgenic plants and products produced by transgenic plants.

33 Crop GM Trait Company Commission Decision

Swede rape Herbicide Tolerance AgrEvo Approved

Maize Herbicide Tolerance AgrEvo Approved

Maize Insect Tolerant Monsanto Approved

Maize Herbicide Tolerance Novartis Approved

Maize Herbicide Tolerance Pioneer Pending

Swede rape Herbicide Tolerance Plant Genetic System· Pending

Fodder Herbicide Tolerance DLF Trifolium, Pending beet Monsanto, Danisco

Cotton Herbicide Tolerance Monsanto Pending

Swede rape Herbicide Tolerance AgrEvo Pending

Cotton Insect Tolerant Monsanto Pending

Potato Modified Starch Amylogene Pending content

Table 9: GM crops in which marketing consent was granted in Europe as of December 1997.

Some transgenic plants have already been developed that produce anti-fungal proteins (AFPs) and enzymes, which degrade fungal cell walls and many others are presently under construction. AFPs which are found principally in seeds, are small, potently antl-fungal compounds that can inhibit the growth of plant pathogenic fungi at concentrations in the range 1-50 parts per million. Anti• fungal chitinase (chitin is the principal fungal cell wall component in the majority of fungi) and ß-l,3-glucanase (ß-l,3-glucan is the principal cell wall polysaccharide in the Oomycetes, an impor• tant group of plant pathogenic fungi) are the two main enzymes which have be Introduced into crop plants.

The principal question with regard to the development and use of transgenic plants producing anti-fungal metabolites is the possibility that such crops can alter the natural microbial popula• tion. Since many microorganisms within the soil are beneficial to the plant and some of which can prove to be essential for normal plant growth, it is vital that the effects of transgenic crops on the native microbial population be assessed.

Within IMPACT this question is being addressed. Specifically the aims of the consortium within this area are to:

1) Investigate perturbations in the populations of potentially beneficial microorganisms in the rhizosphere of transgenic plants expressing various anti-fungal proteins rela• tive to non-transgenic controls.

2) To establish whether plants expressing anti-fungal proteins are still able to form sta• ble mycorrhizal associations with AM fungi.

34 Effect of transgenic plants on the microbial population: In order to assess the impact of transgenic plants on the microbial population it is first of all nec• essary to determine the diversity of the microflora on the roots of plants which have not been transformed (i.e. the wild-type cultivar). Classically the approach for such a study involved the iso• lation and characterization of bacteria on agar plates. Among the disadvantages of this approach is the inability to detect cells that have entered a viable but non-culturable state (i.e. cells that are alive but cannot be grown on agar plates). One solution to this problem is the use of 'culture- independent' techniques based on the PCR amplification of 16S rRNA genes. All bacterial cells contain 16S DNA and the sequence for these genes vary between species. There are regions of the genes, however, which are highly conserved (i.e. they are common among unrelated bacteri• al groups). This allows 16S rRNA genes from extracted soil microbial DNA to be amplified and characterized.

An objective within the IMPACT consortium is to determine the impact of transgenic alfalfa expressing chitinase and lysozyme on microbial diversity in the rhizosphere. The baseline evalu• ation of the microbial diversity of the wild-type alfalfa rhizosphere is currently in progress. The nucleotide sequences of 47 16S rRNA clones has been determined, and indicates the presence of two dominant groups of organisms. Forty clones were allocated to the various subdivisions of the proteobacteria; 5 clones were allocated to the alpha subdivision, 15 clones to the beta sub• division and 20 clones to the gamma subdivision. The five clones from the alpha subgroup belonged to the Agrobacterium/Rhizobium group, whereas 14 clones of the beta subdivision belonged to the Rubrivivax subgroup and 16 clones of the gamma subdivision were allocated to the Pseudomonas subgroup. An additional 16S rRNA gene library was established and analyzed using the ARDRA technique (this technique will be discussed later in the brochure). Results from this analysis confirmed the dominance of two bacterial groups belonging to the Pseudomonas and Rubrivivax subgroups.

The impact of transgenic plants on AM fungi: Among the beneficial microorganisms present within the rhizosphere are the mycorrhizal fungi which form symbiotic relationships with the plant. This relationship is often highly beneficial to the plant, the fungus supplying nutrients and, in some cases, offering protection against plant pathogens. The use of transgenic plants producing anti-fungal metabolites and their possible effects on the ability of the mycorrhizal fungus to successfully established a relationship with the plant is therefore an obvious concern. Results from experiments conducted within the IMPACT consortium have demonstrated that transgenic plants with enhanced resistance to pathogens do not interfere with the symbiotic potential of the plants. Experiments with transgenic tobacco expressing low and high levels of anti-fungal metabolites have demonstrated that seedlings were infected with AM fungi, irrespective of the level of expression (Figure 20).

Field trial evaluation with transgenic plants expressing AFP genes: A field trial has been conducted within the IMPACT consortium to determine whether transgenic oilseed rape, modified for the production of the anti-fungal proteins DmAMPl (from Dahlia merkckii) and AceAMPl (from Allium cepa), has an effect on the biodiversity of the fungal popu• lation and on soil enzyme activities. The technique used for the measurement of fungal biodiver• sity was a molecular approach based on the analysis of the ITS region of the nuclear ribosomal opérons (i.e. the PCR-SSCP technique). The SSCP patterns obtained from the samples from all treatments showed some degree of variation between replicates which reflects the expected nor• mal biodiversity within any soil microbial community. The level of variation between treatments, however, was not significantly different from the inter-replicate variation, indicating that the genet• ic modification of oilseed rape had no detectable affect on the biodiversity within the indigenous

35 30-1

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Wild-Type High Low Expression Expression

Figure 20: The impact of transgenic tobacco expressing low and high levels of antifungal proteins on mycorrhizal formation, 4 and 8 weeks after sowing. Four weeks ( B); eight weeks ( M). Error bars represent standard deviation.

fungal population. With regard to the soil enzyme activities that were tested, significant differ­ ences between non-modified oilseed rape varieties were observed. There was little difference, however, between the enzyme activities In the rhizospheres of the transgenic plants and the non- modified parental plants. Indeed, the major factor influencing the enzyme activities was the soil moisture content. Therefore in this field trial, the differences between soil enzyme activities were not attributable to plant genetic modification, but to environmental variation and to differences In plant variety.

The co-operation between research groups with the breadth of scientific experience present with­ in the IMPACT consortium has enabled field trial evaluations to be conducted in which a range of parameters are effectively monitored. Experiments conducted within the consortium have demon­ strated that genetic modification for production of anti-fungal metabolites does not significantly affect the non-target indigenous microflora.

36 Genetic Technologies for Fingerprinting

Since plants take up nutrients through their root system, the fertility of soil is a major factor which determines plant growth and yield. Soil fertility itself depends on numerous abiotic factors such as soil texture and availability of water, nitrogen, phosphorous, potassium, iron and numerous other nutrients. As well as these abiotic components that influence soil fertility, biological factors including the composition of populations of soil organisms (bacteria, fungi, plant pests and pathogens etc.) exert a major influence.

The most important site of interaction between microorganisms and plants in soil is the rhizos• phere. Here microbial growth is stimulated by the "rhizosphere effect", due to the enhanced amount of nutrients derived from plant roots. It is well known that rhizosphere microbial popula• tions have a major effect on plant growth. These effects can be beneficial, benign or deleterious for the plant. Despite the enormous importance of the rhizosphere for plant growth there is a paucity of knowledge about the structure and function of microbial rhizosphere populations. This is due to the complexity of the habitat and the fact that only a small percentage of the total micro• bial population are accessible for detailed analyses. This lack of accessibility is due to the fact that culture conditions for many of these microbes are unknown. As a consequence the influence of these microbes in plant growth promotion and health have yet to be determined.

Recent progress in the field of molecular biology has produced new techniques that can be uti• lized for the detection of soil microorganisms at the genus (i.e. group) and species level. The Polymerase Chain Reaction (PCR) involves the amplification of genetic material (DNA) so that many copies are produced for analysis. This technique can be used to amplify DNA in general or to amplify DNA from specific soil microorganisms. Within IMPACT, the molecular technique used to detect soil microorganisms at the genus and species level is Amplified Ribosomal DNA Restriction Analysis (ARDRA) and, at the species and sub-species level, Random Amplification of Polymorphic DNA (RAPD). Therefore, both techniques can be applied to examine the composition of microbial populations in the plant rhizosphere.

The Use of ARDRA and RAPD Within IMPACT: The primary application of ARDRA and RAPD which are being refined and utilised as part of the IMPACT programme, is to assess the impact of transgenic plants producing anti-fungal metabo• lites and genetically modified microorganisms on native microbial populations. These techniques can provide a genetic fingerprint of soil microbes and microbial populations that facilitate the detection of shifts in populations. Importantly, these techniques can detect microorganisms which cannot be isolated in the laboratory using classical microbiological techniques (i.e. bacte• ria which have entered the viable but non-culturable, VBNC, state) and therefore, generate a more complete picture of microbial populations in the rhizosphere.

Essentially ARDRA involves the analysis of DNA molecules obtained by amplification of DNA sequences associated with bacterial/fungal ribosomes, the cell structures which play an impor• tant role in the "reading" of genetic material. The DNA that codes for ribosomes is highly con• served within a given species but varies between species.

The first stage for ARDRA analysis of soil microbial populations is the isolation of total bacteri• al/fungal DNA. An area of the DNA (ribosomal 16S rDNA) which is specific to a particular group or species of bacteria/fungi is copied and this segment of DNA is amplified using PCR. The DNA is then cut, using restriction enzymes, into different length pieces which, because they differ in size, can be separated using gel electrophoresis. The pattern produced gives a "fingerprint" for

37 a particular group/species (Figure 21). In order to specifically amplify ribosomal DNA, primers need to be designed. Primers may be defined as small segments of DNA that bind to a specific site and allow the DNA to be copied. A number of primers have been designed for amplification of 16S RNA which represent important species/groups within the rhizosphere.

A Restriction Analysis

End labeled amplification products SSU rRNA | spacer | LSU rRNA ▲ A AA0 3' Primer PCR-Amplification with a primer pair AA A A Fluorescein- c=> Digestion with a restriction enzyme A

• Gel electrophoresis using an automated sequencer • On-line detection of fragments Database • Image analysis

• Grouping of isolates Molecular • Assignment of isolates to taxonomie Taxonomy groups independently of their phenotype • Verification of identifications

Figure 21: ARDRA analysis.

38 Employing this strategy, changes in rhizosphere microbial communities of transgenic plants through the introduction of genetically modified biological control agents, may be recognised and linked to the presence or absence of plant­beneficial (or deleterious) microbes. These shifts may also be linked to plant health and yield.

of Randomly Amplifie Polymorphic DNA Markers

PCR amplification using a single primer % fluorescein ■

end labeled amplification products

gel electrophoresis using an automated sequencer on­line detection of the fragments image analysis Database

_ ­ . . · Population studies MOleCUlãr · Grouping of isolates Twninn a* tne s*rain level I yfJIfiy « Infection epidemiology • Verification if strain identit

Figure 22: RAPD analysis.

39 To increase the level of Information on population changes, in particular the fate of a single species of interest (i.e. a plant pathogen, microbial inoculant etc.), random amplification of poly• morphic DNA (RAPD) is applied. In RAPD analysis, members of a certain species of interest are first isolated from soil using classical microbiological techniques. DNA is obtained from these iso• lates and subjected to PCR amplification using a single non-specific primer. When the amplified DNA is separated by gel electrophoresis, the resulting "fingerprints" indicate the degree of genet• ic diversity for that particular soil species (Figure 22). This method assesses the degree of bio• diversity of rhizosphere microorganisms at the subspecies level, that is, it takes a single micro• bial species and looks at the level of genetic diversity in the soil for that species. This is impor• tant in determining the fate of specific microorganisms that are either beneficial or deleterious to plants such as the effect of transgenic plants on a targeted pathogen.

As a consequence of developing and utilizing these strategies the potential ecological impact and effects of introduced biological control agents and transgenic plants can be appropriately assessed prior to progressing to commercial applications.

The use of RAPD analysis for the determination of pseudomonad biodiversity on the rhi- zoplane and in the rhizosphere of sugarbeet inoculated with P. fluorescens Fl 13: In order to accurately assess the impact of genetically modified biological control agents on microbial biodiversity, it is first necessary to determine the impact of wild-type non-modified strains. A field trial was conducted to determine the impact of the wild-type biocontrol agent Pseudomonas fluorescens F113Rif, on the diversity of indigenous pseudomonads in the rhizos• phere (i.e. soil around the plant root) and rhizoplane (i.e. the root surface) of sugarbeet. The five "soil types" analyzed within the experiment were: soil in which no sugarbeet was grown (i.e. bulk soil), the rhizosphere and rhizoplane of non-inoculated sugarbeet and the rhizosphere and rhizo• plane of sugarbeet inoculated with P. fluorescens F113Rif.

At 19 days after inoculation, P. fluorescens F113Rif had no influence on the number of resident culturable fluorescent pseudomonads at the rhizoplane or in the rhizosphere. Using RAPD analy• sis, the number of distinguishably different pseudomonad strains found in each of the five treat• ments ranged from 66 and 85. Only a small percentage (usually less than 10%) of RAPD profiles observed in a given treatment were also found In another treatment. This, In effect, showed that the pseudomonads differed markedly between treatments. The numbers of RAPD profiles and the level of strain diversity, however, were not significantly different between treatments. Therefore, the presence of the sugarbeet root or inoculation with P. fluorescens F113Rif had no impact on the level of strain diversity of the resident pseudomonad population.

Molecular Based Techniques for Monitoring AM Fungi In order to more accurately assess the effect of genetically modified organisms on mycorrhizal fungi it Is essential to conduct experiments under realistic agronomic situations. This has led to the development of new molecular based techniques which allow the accurate detection and iden• tification of AM fungi in root material taken directly from the field. Regular sampling of field sites enables mycorrhizal biodiversity to be followed across time and different crop species.

To date this work has demonstrated a much-reduced level of fungal diversity in agricultural ecosystems when compared to a relatively undisturbed woodland site (Figure 23). The crop plants are dominated by just two AM groups, regardless of site or crop type. Considering the relatively harsh environment of agricultural soil, i.e. the heavy disturbance of ploughing and the addition of chemical fertilizers, it seems likely that the observations are the result of an ecosystem which can only support the more aggressively colonizing AM fungal species such as Glomus mosseae.

40 Such work has established a baseline for which the impact of genetically modified inoculants can be accurately assessed.

Figure 23: A rank abundance diagram of AM fungal taxa colonizing woodland and arable plants. This diagram shows the reduced number of taxa present in arable crops. The steeper curve of the arable samples demonstrates an increased dominance of one fungal taxon compared to the shallower curve of the woodland samples.

Further Applications Of ARDRA and RAPD: The biotechnological and the pharmaceutical industries are both dependent on the frequent development of new products. Finding new substances to be developed into pharmaceuticals was done in the past by combining new basic chemical compounds with new complex organic chemi• cals and then screening large numbers of combinations for interesting activities. Today, the indus• try's interest in naturally occurring substances is increasing; programmes are set up to isolate new microorganisms for the purpose of detecting new compounds.

PCR, combined with ARDRA and RAPD technologies can significantly benefit the search for new, naturally occurring substances by:

1) Looking at the diversity of uncharacterised isolates: On the basis of fingerprint data it would appear that only members of different groups of bac• teria (rather than different species) are characterized for their ability to produce substances, which could potentially be marketed. Therefore only a fraction of bacteria have been char• acterized for such traits and molecular biology can more accurately Identify new isolates. 2) Looking at the diversity within groups already screened for a certain characteristic: Certain groups of bacteria and fungi are known for their ability to produce compounds of interest. Molecular biology allows such groups to be screened, in more detail, for species with the ability to produce new and important compounds.

41 Besides enhancing screening programmes, DNA fingerprints are ideal to provide a rapid method to check strain collections for mlsidentified isolates. Once fingerprint data have been generated for a significant part of a strain collection, misidentified isolates show up as "not matching". These isolates can then be rechecked for morphological and physiological characteristics to reach a correct identification. Furthermore, methods to differentiate or identify microorganisms at the species and strain level are needed in many other important areas such as epidemiology and food safety, where it is necessary to identify microorganisms fast and correctly and to type them below the species level.

These examples emphasize the great potential for the application of genetic fingerprintingtechnique s utilised within the IMPACT programme, not only for the analysis of changes in microbial populations in the soil as a result of introduced species but also to speed up and enhance the industrial work with microorganisms in general and natural products screening programmes in particular.

42 Executive Summary:

Biotechnology has now become an integral part of the agricultural industry leading to the devel• opment of new crops with traits including resistance to pests and pathogens and microorganisms which can control fungal diseases, stimulate plant growth and provide nitrogen to the plant. With the stringent controls on the use of chemical pesticides and fertilizers and the ongoing environ• mental concern with their application, the use of biotechnology is seen as an important alterna• tive. It is expected that biotechnology will increase productivity in the agricultural sector and will lead to environmentally friendly practices, which will contribute enormously to the goal of sus• tainable agriculture.

Among the questions relating to this technology is the possible effect that genetically modified organisms have on human health and the environment. In order to address such questions the European Commision has funded research within this area to the value of approximately Euro 34M in the last decade. The IMPACT programme is one of the research initiatives funded by the EU. The principal goal of the IMPACT programme is to develop genetically modified microorgan• isms (GMMs) and to determine the effect of GMMs and transgenic plants on the indigenous microbial population. This is of utmost importance since many of the microorganisms present in soil are actually beneficial to the plant, through the protection from pests and pathogens and the supply of essential nutrients. The IMPACT consortium consists of seventeen research and indus• trial centres throughout Europe. This transnational network is of major benefit for it not only allows experiments to be conducted on a large scale with multidlsciplinary collaboration, but also allows field trials with GMMs to be conducted with a range of soil types, crops and climatic con• ditions representitive of the whole of Europe.

The three principal groups of GM microorganisms under investigation within the IMPACT consor• tium are biocontrol agents, biofertilizers and phytostimulators. Biocontrol agents are microor• ganisms, bacteria and fungi, which can benefit plant growth by reducing attack by plant pathogens. The ecological benefits of this strategy are very important, since this could lead to a reduction in the use of chemical fungicides and pesticides, the applications of which have aroused community concerns as to their possible harmful effects on the environment and human health. As demonstrated within the IMPACT consortium, the efficacy of biocontrol agents can be improved by genetic modification for increased anti-microbial compound production. Regardless of their biocontrol activity, however, if such strains have a detrimental impact on beneficial microorganisms in the soil then it is unlikely that they would be considered for commercial use. For this reason the impact of GMMs on native bacterial and fungal populations has been an important objective within the consortium. The numerous microcosm studies, greenhouse exper• iments and field trials that have been conducted within the IMPACT consortium have demon• strated that GMMs have no significant impact on the total microbial population and beneficial components of the microflora compared with wild-type controls. Such a result is important for it paves the way for the use of biocontrol agents at a commercial level leading to the reduced appli• cation, or indeed phasing-out, of environmentally and health hazardous chemical fungicides.

The use of biofertilizers and phytostimulators offer an alternative to the application of chemical• ly produced nitrogen fertilizers to the soil. The application of nitrate to the soil causes serious environmental problems through leakage into surface and ground water and accumulation of nitrogen oxides in the atmosphere. Biofertilizers, however, provide nitrogen directly to the plant in a usable form without any serious threat of leakage. GM biofertilizers (nodule forming Rhizobium strains) have been developed within IMPACT that are highly efficient nitrogen fixers and are highly competitive (i.e. they can form symbiotic relationships with plants more efficiently than

43 wild-type strains). The colonization ability of these strains have been confirmed within the con• sortium and impact studies suggest that such strains are suitable for field trial release. Planned field trials that conform rigidly to the EU directive 90/220/EEC are underway. The collaborative aspect of the IMPACT consortium allows a range of parameters to be investigated; in addition to the nodulation capabilities of these strains, their impact on the indigenous microflora including beneficial microbial populations and their effect on crop yield is being assessed.

Phytostimulators can directly promote plant root growth by the production of plant growth stimu• lating factors. On an environmental level, increased plant root growth leads to an enhanced abil• ity to take up nitrogen, resulting in a reduced need for nitrogen fertilizer application and therefore prevent pollution of waterways and ground water. Genetically modified phytostimulators (Azospirillum strains) have been developed within the IMPACT consortium that can effectively pro• mote plant growth and which have no apparent impact on the indigenous microbial population. In addition, inoculation with Azospirillum strains allowed the application of lower levels of nitrogen fertilizer to obtain the same grain yield. GM Azospirillum strains have been produced which have altered growth factor production capabilities and their effect on plant yield, nitrogen uptake and impact on the Indigenous microbial population is presently under investigation. The results from field trials investigating both the biofertilizers and phytostimulators will provide valuable informa• tion on the efficacy of these strains and their effect on native microbial populations under field conditions. There is the need for comprehensive assessment of the efficacy and impact of biofer• tilizers and phytostimulators since the farmer requires a product which is as effective as chemi• cal fertilizers and the public demands strategies which are environmentally friendly.

As with GM biocontrol strains producing anti-fungal metabolites, there is much interest within the IMPACT consortium pertaining to the impact that transgenic plants producing anti-fungal proteins and enzymes have on the native microbial population, with particular reference to the beneficial microflora. Experiments conducted within the IMPACT consortium have demonstrated that modi• fication of tobacco for anti-fungal protein production did not interfere with the symbiotic relation• ship between the plant and AM fungi. This is important since, even in soils in which fungal pathogens are present, if the production of anti-fungal proteins had a detrimental effect on AM fungi, then the disadvantages of the genetic modification could outweigh the benefits. In order to determine the impact of transgenic plants on microbial populations, it is first of all necessary to determine the effect of wild-type plants. Transgenic alfalfa plants, genetically modified for chiti- nase and lysozyme production, are currently being engineered within the IMPACT consortium and their impact on microbial populations will be compared with the baseline scientific information obtained for wild-type alfalfa. The multidisciplinary structure of the IMPACT consortium allows a great deal of information to be obtained from the planned field trials with transgenic plants. In addition to determining the effect that transgenic plants have on target pathogens, the impact that the transgenic rhizosphere has on the indigenous microflora, using classical microbiological the molecular techniques, will be assessed and compared with the impact of the wild-type plant cultivar.

The IMPACT project has been funded by the European Commision in the framework of the EC biotechnology programme (Framework Programmes III and IV). New knowledge has been accu• mulated regarding the fate of genetically modified microorganisms in soil systems and their impact on native microbial populations. It is only with field release studies, however, that rele• vant Information can be obtained pertaining to the activity of GMOs in commercial agricultural sys• tems. Some of the first field trials conducted under the guidelines specified within the EU direc• tive 90/220/EEC for deliberate release of GMOs, were carried out within the IMPACT consortium. The breadth of expertise within the consortium allowed many parameters to be investigated

44 which could not have been achieved without the multldisciplinary and transnational approach adopted. This aspect is the key strength of the IMPACT project. It will facilitate ongoing research into the use of novel microorganisms and plants engineered with appropriate traits to compete effectively with chemical pesticides, fungicides and fertilizers and, in the future, to replace such chemicals to achieve the goal of environmentally friendly, sustainable agriculture.

Conclusions:

• Modern biotechnology encompasses many issues ranging from the improvement of human health and the protection of the environment, to other social aspects covering employment and economic factors. It is clear that biotechnology has emerged as one of the most promising and crucial technologies for substantial development in the next century. Looking to the future it is expected that given improvements in the business environment, biotechnology activity by the year 2005 will have reached a value of approximately Euro 250 billion and will have created more than 3 million jobs in the EU.

• The agri-food sector has been highlighted as a key area where substantial growth in biotech• nology activity will occur. This is not surprising in view of the stark reality that over the next 30 years food requirements for the global population will exceed the total amount of the food pro• duced over the past 10,000 years. Already modern biotechnology is revolutionising the agri- food sector. It is leading to the development of novel crops genetically modified for character• istics including resistance to pests and pathogens and new environmentally friendly microbial inoculants to protect plants from disease and increase plant growth. These new products are expected to lead to a reduction in the use of pesticides, fungicides and fertilizers and provide new options for the control of crop diseases which currently cannot be managed even with existing agri-chemicals.

• As with all new technologies it is natural that a number of Issues concerning the development and use of genetically modified organisms in agriculture are addressed. The main issues cen• tre largely on biosafety matters particularly regarding the long-term effects that GMOs may pose to human health and the environment. As a precautionary measure, the EU and several of its member states have supported biosafety research to gain experience with the safety aspects related to the release of GMOs as well as to underpin in a scientific way the regula• tory framework and activities concerned.

• In the framework of the European Community's biotechnology programme a significant portion of research effort has been devoted to the evaluation of environmental risks associated with the deliberate releases of GMOs into the environment. Approximately 4% of the budgets of the EC biotechnology programme has been allocated to biosafety research (Euro 42.6M) since 1987. The IMPACT project involving 17 research and industrial centres of excellence in 8 European countries is one example of a multidisciplinary network that was established and funded under the biosafety research and development framework of the EU biotechnology pro• gramme. The IMPACT programme deals with the interactions between microbial Inoculants and resident populations in the rhizosphere of agronomically important crops in typical soils.

• The general research priorities set in the IMPACT project were the study of biological phenom• ena related to the release release of GM plants and microbial inoculants into the environment. Technology and know-how underpinning biosafety and microbial ecology research has been developed in the IMPACT project and is helping provide the necessary scientific supports for

45 the different regulations required for the harmonious development of biotechnology.

The IMPACT project has provided an important biosafety demonstration component for the release of GMOs. Specifically a number of GM microbial inoculants with relevance for envi• ronmentally friendly food production have been released and tested under commercial field conditions in a number of European countries under the terms of the EU Directive 90/220/EEC for the deliberate release of GMOs. Data from field experiments indicate the lack of significant detrimental effects associated with the use of wild-type or genetically modified microbial inoculants on crop yield, soil biomass, soil fertility and selected soil microorganisms, including taxonomically similar strains and beneficial soil microorganisms. For example, the impact of a genetically modified biological control agent on soil enzyme activities (as a mea• sure of biological perturbations in the rhizosphere) is far less than the impact observed with the conventional use of commercial fungicide treatments for disease control in agriculture.

The IMPACT project is providing a transnational network that is contributing synergy on biosafe• ty, risk, ecological and environmental principles. Training and mobility schemes addressing key issues for biosafety and microbial ecology R+D have been initiated and developed. The EU backed project provides complementary biosafety research efforts to national research initia• tives in member states. However the research effort significantly benefits from the added value of international co-operation in providing a critical mass of expertise in multidisciplinary areas that underpin modern approaches and strategies to biosafety issues.

46 Achievements of the IMPACT Programme and Future Directions:

1) Within the IMPACT consortium, modern biotechnological techniques have been utilized to develop new strains of beneficial microorganisms which have been improved by genetic modification. These include:

GM Biological Control Agents - Pseudomonas strains, genetically modified for enhanced anti-fungal metabolite production, showing increased control of plant dis• eases.

GM Phytostimulators - Azosprillum strains with enhanced production of plant growth stimulating factors, showing increased plant root growth resulting in increased uptake of nitrogen.

GM Biofertilizers - Rhizobium strains with high nitrogen fixation efficiency, genetically mod• ified for enhanced nodulation competitiveness, supplying nitrogen directly to the plant.

2) Microbial inoculants have been developed that can be monitored under natural agricultural conditions. The marking systems utilized do not rely on the introduction of antibiotic resis• tance genes, which could lead to undesirable antibiotic resistance In nature, but involve the insertion of genes which do not confer any ecological advantage (e.g. lacZY, gusA).

3) The use of GMMs have been shown to be environmentally friendly alternatives to chemical applications. GM biological control agents have been shown to be as effective as fungicide treatments and GM phytostimulators and GM bioferilizers reduce the need for the applica• tion of nitrogen fertilizers.

4) An assessment has been made of the environmental impact of GMMs inoculated into soil. Evidence suggests that impact is minimal and substantially lower than the use of chemical applications. GMMs have been shown to have no detrimental effect on the beneficial frac• tion of the soil microflora, including AM fungi and ingigenous Rhizobium populations.

Against the background of significant progress already achieved within the IMPACT consortium, additional R+D efforts are required to further develop the safety aspects related to the release of GMOs as well as to underpin the scientific basis for regulatory frameworks. Among the areas which require further investigation are:

1) Fundamental research pertaining to the population structure and dynamics of microorgan• isms in the rhizosphere.

2) The signals, traits, and associated mechanisms influencing microbial behaviour in different environments.

3) Additional factors which accurately reflect the impact of introduced microorganisms and transgenic crops and their residues on ecosystems.

4) The development of new molecular-based systems for the detection and monitoring of GMMs released into the environment.

5) The development of biological containment systems which prevent the transfer of genetic material between microorganisms.

47 ANNEX Ί

The IMPACT Consortium

Co-ordinator PROF. F. O'GARA, Biomerit Research Centre, Microbiology Department, National University of Ireland, Cork, Ireland. Tel. +353-21-272097 Fax. +353-21-275934 E-mail: [email protected] Research Activities: The development of GM biocontrol agents; molecular microbial ecology of the rhizosphere; impact of biocontrol agents on indigenous rhizosphere microorganisms; the VBNC state in Rhizobium.

Partners PROF. J.M. BAREA NAVARRO, CSIC, Estación Experimental del Zaidin, Profesor Albareda 1, 18008 Granada, Spain. Tel. +34-58-121011 Fax. +34-58-129600 E-mail: [email protected] Research Activities: The impact of biological control agents, biofertilizers and phytostimulators on arbuscular mycor• rhizal formation.

PROF. P. BONFANTE, Dipartimento di Biologia Vegetale, Università di Torino, Viale P.A. Mattioli 25, 10125 Torino, Italy. Tel: +39-11-6699884 Fax: +39-11-6707459 E-mail: [email protected] Research Activities: Interactions between rhizosphere saprotrophic and mycorrhizal fungi; Impact of GM bacterial strains and transgenic plants on saprotrophic and mycorrhizal fungi.

PROF. SERGIO CASELLA, Dipartimento di Biotecnologie Agrarie, Agripolis - Università di Padova, Strada Romea, 16, 35020 Legnaro, Padova, Italy. Tel: +39 49 8272922 / 8272905 Fax: +39 49 8272929 E-mail: [email protected] Research Activities: Assessment of the fate of microbial Inoculants released in the field with particular reference to the VBNC state in Rhizobium; impact of GMMs on microbial populations in field conditions (in collaboration with Agronomica).

48 ANNEX Ί

PROF. G. DÉFAGO, Institute of Plant Sciences/Phytomedicine, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland. Tel: +41-1-6323869 Fax: +41-1-6321108 E-mail: [email protected] Research Activities: Impact of wild type and GM strains on the indigenous rhizosphere population; the VBNC state in Pseudomonas; the fate of wild type and GM biocontrol strains released into the environment.

DR. ANDY GREENLAND, ZENECA Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6ET, U.K. Tel. +44 0 1344 414820 Fax. +44 0 1344 414996 E-mail: [email protected] Research Activities: Provision of materials and collaboration on the impact of transgenic plants, expressing antifun• gal proteins and enzymes, on the indigenous rhizosphere microflora.

MR. D. GROGAN AND J. BRODERICK, Irish Sugar pic, Athy Road, Carlow, Ireland. Tel: +353-503-31708 Fax: +353-503-43087. E-mail: [email protected] / [email protected] Research Activities: Bacterial biological control agents for sugarbeet diseases; colonization ability of biological con• trol agents on the roots of sugarbeet, under field conditions.

PROF. D. HAAS, Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland. Tel: +41-21-6925631 Fax: +41-21-6925635 E-mail: [email protected] Research Activities: Characterization of Pseudomonas strains as biological control agents and their improvement by genetic engineering.

PROF. E.J.J. LUGTENBERG, Institute of Molecular Plant Sciences, Cluslus Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, P.O. Box 9505, 2300 RA Leiden, The Netherlands. Tel: +31-71-5275065 Fax: +31-71-5275088 E-mail: [email protected] Research Activities: Isolation and characterization of Bacillus biocontrol strains; impact of transgenic plant rhizos• phere on beneficial gram positive bacteria.

49 ANNEX Ί

PROF J.M. LYNCH, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, U.K. Tel: +44-1483-259721 Fax: +44-1483-259728/259396 E-mail: [email protected] Research Activities: Assessment of the effect of GM biological control agents on soil enzyme activities; isolation and characterization of Trichoderma biocontrol strains; nematodes as a factor in rhizosphere function.

PROF. M.P. NUTI, Dipartimento di Chimica e Biotecnologie Agrarie, Università di Pisa, Via del Borghetto, 80, 56124 PISA, Italy. Tel: +39-50-578640 Fax: +39-50-571562 E-mail: [email protected] Research Activities: Assessment of the fate of microbial inoculants released in the field with particular reference to the VBNC state in Rhizobium..

PROF. J. OLIVARES, CSIC, Estación Experimental del Zaldin, Profesor Albareda 1, 18008 Granada, Spain. Tel: +34-58-121011 Fax: +34-58-129600 E-mail: [email protected] Research Activities: Development of GM Rhizobium strains with enhanced nodulation competiveness; assessment of interactions between GM strains in field conditions.

DR. U. PERUCH, Agronomica S.r.l. Consortile, Via Romolo Gessi, 20, 48100 Ravenna, Italy. Tel: +39-544-35078 Fax: +39-544-36589 E-mail: [email protected] Research Activities: Agro-ecological evaluation of phytostimulators and biocontrol agents under field conditions; effect of GM bicontrol agents on specific pathogenic fungi and the indigenous microflora.

PROF. A. PÜHLER, Biologie VI (Genetik), Fakultät für Biologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany. Tel: +49-521-1065607 Fax: +49-521-1065626 E-mail: puehler@genetik. uni-bielefeld.de Research Activities: Assessment of the structure and function of the rhizosphere in crop plants; utilization of PCR- based methods for the comparative analysis of the genetic diversity of microbial populations in the rhizospheres of wild-type and transgenic crops.

50 ANNEX Ί

DR. T. RUIZ-ARGÜESO, Departamento de BiotecnologiaE.T.S. de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Cuidad Universitaria, s/n E28040 Madrid, Spain. Tel: ++34-1-3365752/59752 Fax: +34-1-3365757 E-mail: [email protected] Research Activities: Assessment of Chemotaxis with particular reference to methyl accepting chemotactic proteins for targeting Rhizobium to the pea rhizosphere; analysis of the thyA containment system.

DR. R.J. SCHEFFER, Novartis Seeds B.V., P.O. Box 21600 AA Enkhuizen, The Netherlands. Tel: +31-228-366235 Fax: +31-228-366348/312818 E-mail: [email protected] Research Activities: Control of phytopathogens and pests in agricultural and horticultural systems with a focus on veg• etables; formulation and delivery systems of seed-applied crop protection agents, of chemical or biological origin.

DR. R. SIMON, TÜV Energie- und Systemtechnik GmbH, ISB, Engesserstrasse 4b, 79108 Freiburg, Germany. Tel: +49-761-5597-341 Fax: +49-761-5597-349 E-mail: [email protected] Research Activities: Genetic fingerprinting of rhizosphere microorganisms using ARDRA analysis of DNA directly extracted from soil for the assessment of the impact of GM inoculants on the indigenous micro• bial population.

PROF. J. VANDERLEYDEN, Katholieke Universiteit Leuven, F.A. Janssens Lab. for Genetics, K.U. Leuven, Faculty of Agri and App Biological Sci, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. Tel: +32-16-329679 Fax: +32-16-321966 E-mail: [email protected] Research Activities: Development of GM Azospirillum strains with altered growth factor production and assessment of plant growth stimulation and nlrogen uptake.

PROF. J.P.W. YOUNG, Department of Biology, University of York, PO Box 373, York Y010 5YW, U.K. Tel: +44-1904-432914 Fax: +44-1904-432860 E-mail: [email protected] Research Activities: The study of the diversity of AM fungi at the molecular level; the impact of GM inoculants on AM fungi.

51 ANNEX Ί

Science Panel Members PROF. M. NUTI (Chairman), Dr. I. ECONOMIDIS, PROF. P. YOUNG, PROF. J. LYNCH, PROF. F. O'GARA.

EU Biotechnology Project Officer DR. I. ECONOMIDIS, DG RESEARCH, Commission of the European Communities, Rue de la Loi 200, B-1049 Brussels, Belgium. Tel: +32-2-2951574 Fax: ++32-2-2991860 E-mail: [email protected]

Financial Administration MR. M.F. KELLEHER/MS. M. HEALY, Finance Office, University College, Cork, Ireland. Tel: +353-21-276871 Ext.2347 Fax: +353-21-275948 Email: [email protected]

This brochure was compiled by Dr Uitan Walsh and Professor Fergal O'Gara (Biomerit Research Centre, National University of Ireland, Cork) and Dr. loannis Economidis and Stéphane Hogan of the European Commission, with the assistance of the partners of the IMPACT consortium.

52

Harnessing the potential of genetically modified microorganisms and plants.

Luxembourg: Office for Official Publications of the European Communities

1999 — 52 pp. — 21 χ 29.7 cm

ISBN: 92-894-0295-4

Venta · Salg · Verkauf · Πωλήσεις · Sales · Vente · Vendita · Verkoop · Venda · Myynti · Försäljning

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