Establishing the Efficacy, Safety and Persistence of biopesticides based on naturally occurring beneficial bacteria

HARNESSING NATURAL BACTERIA TO PROTECT CROPS AND SUSTAIN AGRICULTURE
The aim of our research is to establish the fundamental scientific knowledge required to use naturally occuring plant protective bacteria in an effective, safe and environmentally non-persistent manner. Current agriculture is reliant on the use of chemical pesticides to protect crops from attack by disease-causing microorganisms. One such disease, known as damping off, is a major problem killing crop plants as they germinate. To feed the growing world population we will need to intensify agriculture and adding more chemical pesticides to protect crops. We cannot sustain high usage of pesticides, especially when they damage the environment and human health. Alternative approaches which are sustainable must be developed and natural bacterial biopesticides offer an attractive solution to enhance future agriculture and protect crops from disease.

BURKHOLDERIA AMBIFARIA - A NATURALLY PLANT PROTECTIVE BACTERIA
Several natural bacteria live very closely with plants and provide protection against disease causing microbes. One group of bacteria, Burkholderia ambifaria (B. ambifaria), when applied as a seed coat are very good at protecting germinating crops from lethal attack by damping off fungi. This led to their safe use as biopesticides up to 1999 in the US, but during the 1990s, a related group of Burkholderia bacteria were found to cause lung infections in people with cystic fibrosis. At the time, the safety of B. ambifaria could not be fully defined, it was decided that registration of new biopesticides would not be permitted until they were demonstrated to have no health risks. Although the registered Burkholderia biopesticide products could still be used, the agricultural industry moved away from using them and expanded their use of chemical pesticides to sustain agriculture.

UNDERSTANDING HOW BURKHOLDERIA AMBIFARIA PROTECTS PLANTS AND ENSURING IT IS SAFE AND ENVIRONMENTALLY NON-PERSISTENT
Using modern research techniques, we found B. ambifaria's plant protective ability was due to a set of genes that the bacterium uses to make an antifungal molecule called cepacin. When the cepacin-making genes were removed, plant protection was lost from the bacterial seed coat. By sequencing all the genes in B. ambifaria bacteria we also identified multiple new genes that make natural products potentially exploitable as biopesticide molecules, antibiotics or fine chemicals. These new findings have allowed us to map out a research program to re-purpose B. ambifaria as a biopesticide which will be carried out as the following four research objectives:

1. We will map all the molecules and pathways B. ambifaria uses to protect plants from attack by pathogens, specifically identifying those which are switched on when the bacteria grow on the exudates released by germinating crop seeds.

2. We will engineer the cepacin pathway allowing it to be moved into other biotechnological bacteria and also exploring how it is able to make the unusual chemical structure of this biopesticidal molecule.

3. We will improve the safety of B. ambifaria by identifying the genes it uses to cause disease and exploring different strategies to remove these genes. Infectious live bacteria can be used as vaccines when specific genes are removed, and the same approach will be exploited to make a safe biopesticide.

4. We will explore what happens to B. ambifaria when it is placed in the soil as a seed coat and how it affects other soil microorganisms, so that we understand it's environmental persistence.

Overall this integrated research programme will seek to deliver an effective, safe and environmentally non-persistent B. ambifaria biopesticide, which can significantly enhance and sustain agricultural production without the need to use chemical pesticides.

OVERALL AIM

There is an urgent unmet need to reduce chemical pesticides use and develop alternative approaches to sustain agricultural production. We will obtain the fundamental biological data to enable effective, safe and environmentally non-persistent exploitation of natural bacterial biopesticides, specifically repurposing Burkholderia ambifaria as a historically effective agent.

UNDERPINNING DATA AND APPROACHES

Genome mining of a unique collection of 64 B. ambifaria defined 7 known antimicrobial metabolites and >30 interesting secondary metabolite pathways. A novel pathway encoding biosynthesis of the polyyne antifungal, cepacin, was identified and shown to mediate protection of peas against damping off by the plant pathogen Pythium, when B. ambifaria was applied as a seed coat. Deletion of the B. ambifaria third genomic replicon, which results in reduced virulence, left the damping off control phenotype intact. From this knowledge base, we will apply an interdisciplinary genomics, chemical biology, crop-interaction assays and soil microbiome analyses to repurpose B. ambifaria as a biopesticide.

OUR HYPOTHESIS is "Pathogen targeted, virulence-attenuated Burkholderia biopesticides are effective, safe and environmentally non-persistent,"

TECHNICAL OBJECTIVES

1 - EFFICACY MAPPING: To define the biosynthetic pathways, activation signals and metabolites that underpin B. ambifaria biopesticidal activity of against multiple crop pathogens.

2 - EFFICACY ENGINEERING: To harness Burkholderia polyynes as potent, environmentally non-persistent biological control molecules.

3 - SAFETY: Using transposon-sequencing and third replicon deletion as next generation tools to map beneficial biopesticidal genes and in parallel identify pathogenic pathways.

4 - PERSISTENCE: To apply culture and culture-independent analysis to understand the persistence of B. ambifaria biopesticides with the soil microbiome.

Our research on bacterial biopesticides will impact on multiple beneficiaries as follows:

1. GLOBAL FOOD SECURITY AND SUSTAINING AGRICULTURAL PRODUCTION
To feed the world's expanding population, agricultural production will have to intensify. Global crop production is dependent on the application of chemical pesticides, however, their impact on health and the environment is an increasing challenge. For example, thiram, a widely used antifungal seed treatment used to prevent damping off disease in germinating crops, will be withdrawn from the Europe this year. Pathogen resistance to historical chemical pesticides is also a problem. Overall, there is an urgent need to develop novel pesticide approaches, and by exploiting naturally protective bacteria such as Burkholderia that have coevolved with plants, we can develop biopesticides for sustainable agriculture.

2. COMMERCIAL EXPLOITATION BY SEED COAT COMPANIES
The global market for chemical pesticides is substantial with the US spending greater than $500 million p.a. on fungicides to maintain their agricultural production. Up to 80% of crop losses may occur immediately after planting as germinating plants are highly vulnerable to pathogenic bacteria, fungi and other eukaryotic pests. Damping off disease, where fungi or fungal-like species attack germinating crops is a major global problem, globally worth greater than $288 million per annum. The application of seed coats to crops has multiple agricultural benefits such as the ability to incorporate nodule forming bacteria (rhizobia) to benefit nitrogen fixing leguminous crops. The incorporation of biopesticidal bacteria into seed coats can deliver crop protection against plant pathogens. We have begun discussions with the seed coat company, Centor Oceania, about the potential for incorporating B. ambifaria biopesticides into their products (see support letter). Continuing industry discussions and our "Biopesticides for sustainable agriculture" impact meeting, will seek to commercially translate our research. Also by exploitation of Burkholderia bacteria as a novel source for natural products, we can increase the bank of bioactive molecules available to the agrochemical and pharmaceuticals industry.

3. GOVERNMENT AND ENVIRONMENTAL REGULATORY BODIES
Food security and sustaining agriculture is a key government priority. The Department for Environment, Food and Rural Affairs (DEFRA) is charged with delivering policies and bodies such as the Environment Agency regulating the use of pesticides in agriculture. The systematic interdisciplinary approach we are undertaking to re-purpose Burkholderia as a biopesticide will illustrate to government and regulatory bodies how cutting-edge biotechnological strategies can overcome multiple current issues in agriculture. To disseminate our research, we will involve these regulatory government bodies in our impact meeting (see impact plan).

4. THE PUBLIC AND THE NEED TO SUSTAIN AGRICULTURE
The UK is fortunate to have a high standard of living where the impact of food shortages has not been widely felt. However, the public need to be informed of the global food security issues that are a major challenge. Our research provides a natural strategy to reduce use of toxic and persistent chemical pesticides, and we will engage the public in biopesticide research and other strategies for sustainable agriculture via outreach events with children and an open day discussion as a two-way communication to inform our research direction (see impact plan).

5. TRAINING OF HIGHLY-QUALIFIED INDIVIDUALS IN INTERDISCIPLINARY BIOSCIENCES RESEARCH
Our research team brings together experts in microbiology, genomics, natural product chemistry and plant sciences science, to implement an interdisciplinary research programme on bacterial biopesticides. We will equip the early career researchers recruited on the grant with skills that are vital for the future of UK academia and industrial excellence.