David Ritchie

The treatment of plant pathogens with traditional antimicrobials drives the evolution of resistance among pathogens in two ways: 1) selection favoring resistance in the pathogen population, and 2) selection favoring resistance genes in the microbial community. Since bacterial species readily exchange genes, resistance genes from the community represent a major source of resistance. Moreover, removal of community members eases the route of invasion for pathogens. This suggestion aims to circumvent antimicrobial resistance by targeting the pathogens specifically. That is, this suggestion will support the development of customized targeted antimicrobials that will kill specific pathogens yet leave beneficial microbial flora unharmed. As a result these antimicrobials won't contribute to the spread of antibacterial resistance or disrupt beneficial microbial communities. This proposal builds on our previous work targeting Citrus pathogens and, in effect, tricks the pathogen into recognizing its genome as a foreign invader through the use of an engineered CRISPR system. The use of the sequence specific recognition results in unparalleled specificity in antimicrobial action. We propose to build on progress made in a previous project focused on Candidatus Liberibacter asiaticus. Development of that system was validated in the more tractable Xanthomonas citri disease system and as a result, we developed a CRISPR-antimicrobial (CRAM) construct to target the Xanthomonas citri genome. Many of the barriers to this technology were bypassed by its application to a culturable pathogen. Here, we propose to complete the CRAM construction to treatment of X. citri by amending the system to a bacteriophage genome capable of delivering this construct to X. citri. Moreover, we propose to utilize this scheme to build CRISPR-antimicrobial constructs for three additional bacterial plant pathogens of regional and national importance : Pseudomonas syringae, Xathomonas arboricola pv. pruni and Ralstonia solanacearum. These bacteria act as both an endpoint demonstrate of the pipeline developed previous as well as a starting point for the development of augmented bacteriophage both as a means of pathogen control and in vivo engineering. Our approach or pipeline involves the isolation of bacteriophage delivery vehicles, development of genetic systems, and the incorporation of CRISPR antimicrobial constructs geared toward specific, programmed killing of each target pathogen. Finally, we propose to investigate the use of an avirulent Ralstonia strain for the in situ amplification of the engineered bacteriophage for improved control of the pathogenic strain. Such an approach may provide a route for the development of soil inocula to provide extended suppression of, or even protection from invasion of, Ralstonia.

North Carolina State University (Cooperator) and the Agricultural research Service (ARS or Agency) desire to enter into this Agreement for the purpose of supporting research to be carried out at ARS and Cooperator facilities. ARS desires the Cooperator to provide goods and services necessary to carry out research of mutual interest within Raleigh, North Carolina.

Fungicides provided by BASF will be evaluated for control of the fungal disease brown rot of peaches. The protocol will be as set out in number DEV-F-2015-US-GJK-A-01.0 as provided to the Project Coordinator.

Evaluation of materials for management of bacterial spot on stone fruits (peaches) will be evaluated.

Yellowing and elongation of grass tillers, called etiolation, has been a nuisance in turfgrasses for many years, with numerous observations around the world. Many suspected causes of this problem have been named, including fungi, viruses, phytoplasmas, or nutrient deficiencies. Over the last 10 years, etiolation seems to have become more common on creeping bentgrass putting greens. In most locations, the symptoms are transient, coming and going with changes in weather conditions. In other locations, however, it has become a chronic problem and leads to gradual thinning and death of affected turf during summer or other times of stress.

1. Identification and Significance of the Innovation Each year the Agriculture Industry loses hundreds of millions of dollars in produce due to bacterial plant diseases. Biocides, most of which are copper-based, are employed to combat these pathogens. Currently, millions of pounds of copper are sprayed on our produce annually, and as a result, the Agriculture Industry is increasingly being confronted with copper-resistant strains of pathogenic bacteria. Additionally, serious toxic effects have been reported as a direct result of copper being introduced into our soil and water supply. In order to protect our environment from the effect of copper toxicity and to overcome increasing copper-resistance of pathogens, novel strategies must be developed to reduce usage of copper biocides and to more effectively treat agricultural pathogens. Agile Sciences has developed a unique approach to reducing the Agriculture Industry?s reliance on copper-based biocides. Agile Sciences? co-founders have shown that a novel series of compounds, derived from a marine natural product that protects sea sponges from bacterial colonization (?biofouling?), are able to effectively remove bacteria from their protective biofilm state so that the bacteria are >10 times more susceptible to copper treatments. Furthermore, these unique compounds, called ?Agilyte??, are able to disable the resistance mechanisms of copper-resistant strains of bacteria so that these strains become susceptible to the copper treatment. The scope of this Phase I proposed work is to synthesize Agilyte? molecules that will work synergistically with low rates of copper to control the common bacterial pathogen Xanthomonas sp. Co-administering an Agilyte? molecule with copper is expected to substantially lower the quantities of copper needed to achieve disease control, providing a more effective and environmentally desirable product.

he agriculture industry suffers tremendous crop loss due to bacterial infections. It has been estimated that these losses approach $40 billion annually. Current techniques for controlling bacterial infection of crops include direct treatment by antimicrobial chemicals, disinfectants, and copper sprays or indirectly through insecticides to eliminate pests who serve as vectors for the diseases. All of these methods carry risks of environmental contamination. Concern is also present regarding the use of antibiotics, which may remain present in food and contribute toward the development of resistant bacteria dangerous to humans. Furthermore, many bacteria are resistant to all current forms of antibiotic treatment. Bacteria that infect plants are extremely resistant to eradication because they form biofilms. Biofilms are defined as a surface attached community of bacteria that are protected by an extracellular matrix of biomolecules. Biofilms are inherently insensitive to disinfectants and antibiotics. For example, it has been estimated that bacteria residing within the biofilm state are 1,000-10,000 times more resistant to antibiotic treatment. Given the prevalence of bacterial biofilms that infect the various produce in both North Carolina and the rest of the world, the development of non-toxic methods to control and/or eradicate bacterial biofilms would have a tremendous impact on both economics within our state and allow us to make fundamental advancements in the ability to treat plant disease. At North Carolina State University, we have developed the only class of non-toxic small molecules that inhibit and disperse bacterial biofilms across both order and phylum. To date, we have a 100% success rate of inhibiting and dispersing biofilms against all bacterial strains we have studied. A majority of the strains we have studied are medically relevant ã-proteobacteria, including Pseudomonas aeruginosa and Acinetobacter baumannii. The major pathogens that destroy crops are also ã-proteobacteria, especially of the genus Xanthomonas. Based upon our tremendous success in inhibiting and dispersing biofilms with non-toxic small molecules, we hypothesize that some of our molecules will find use in the agriculture industry as the next generation agents to treat bacterial disease. Therefore, the Research Aims of this proposal are: Research Aim 1: Screening of our small molecule library to identify hits against Xanthomonas strains. Research Aim 2: Conduct analogue synthesis on identified hits to tune activity against Xanthomonas strains. Research Aim 3: Conduct both in vitro and field trials to assay compound efficacy. Research Aim 4: Toxicity and mechanism of action studies.