This Small Business Innovation Research Phase I project will generate a "light-tagged" reporter phage that can confer a bioluminescent signal to the phytopathogen Pseudomonas syringae pv. alisalensis. P. syringae pv. alisalensis is the causative agent of bacterial blight, a contagious and damaging disease afflicting cruciferous vegetables. Since the severity of the disease renders the crop unmarketable, it is essential to be able to correctly identify the agent on asymptomatic and diseased plants, and from inoculum reservoirs. The Phase I goal is to develop a bioluminescent reporter phage for the detection of P. syringae pv. alisalensis. The objective is to generate a specific P. syringae pv. alisalensis light-tagged reporter phage and then perform feasibility studies to demonstrate that the light-tagged reporter can be used as a P. syringae pv. alisalensis detection system. The research is expected to generate a viable recombinant reporter phage that can rapidly, sensitively, and specifically confer a bioluminescent signal response, and hence detect P. syringae pv. alisalensis.
The broader impact/commercial potential of this project is that the P. syringae pv. alisalensis diagnostic may help to reduce damage to commercially grown vegetable crops throughout the U.S. Symptoms first appear as small water-soaked flecks on the lower foliage. These flecks or lesions expand and become surrounded by bright yellow borders that eventually coalesce to form large necrotic areas. Bacterial blight infestations can result in crop damage to 60% of the commercial field. P. syringae pv. alisalensis has a broad host range afflicting monocots (California brome, oats), crucifers (cauliflower, broccoli, broccoli raab, brussels sprouts, cabbage, radish, arugula) and tomato. The value of Brassica oleracea vegetables alone (e.g. broccoli, cauliflower, cabbage and brussels sprouts) is estimated at $1.3 billion annually in the U.S. Consequently, the economic losses due to bacterial blight are potentially severe. However, to date, there are no commercially available or approved P. syringae pv. alisalensis detection methodologies. The long-term goal of this research is to develop a novel diagnostic kit for the detection of this agriculturally important phytopathogen. The research also may provide the foundation technology for the development of biosensors for other agriculturally important bacterial pathogens.
Pseudomonas cannabina pv. alisalensis is a bacterial phytopathogen which means it causes disease on plants. The plants this bacterial pathogen infects and causes disease on are important vegetables crops such as broccoli, cauliflower, Brussels sprouts, and cabbage. If the vegetable field is infected with the pathogen, the pathogen eats away at the crop, rendering the crop unmarketable, and not fit for human consumption. As the disease can be passed from field to field, and because it is possible to treat the disease assuming it is identified early during infection, there is value in developing a sensor which can detect the presence of this pathogen. The goal of this research was to generate a sensor which can detect the presence of P. cannabina pv. alisalensis as this bacterial pathogen causes disease on agriculturally important crops. To accomplish this goal, bacteriophage were genetically engineered to create bioluminescing reporter phage. Bacteriophage are naturally occurring viruses which only infect specific bacteria, such as P. cannabina pv. alisalensis. Genetic engineering was utilized to create reporter phage, which in the presence of the target bacteria, caused the bacterial pathogen to give off a light signal which could be easily measured. If the bacterium was not present, however no light signal was produced. Thus, this test can be used to see if the pathogen was present or absent as indicated by the presence or absence of a light signal, and be used to determine if the plant is infected with the disease. Thus, the technology displays promise as a bacterial diagnostic for this agriculturally important disease.
