PathSensors, Inc. — Department of Agriculture SBIR Phase I: 8.13

Description

Bacterial wilt is a disease that occurs in many important agricultural crops, including pepper, tobacco, tomato, and potato, and can be caused by the bacterium Ralstonia solancearum. This bacterium is classified into 5 races that are based loosely on host ranges, and into 5 biovars, which are based upon their differential ability to utilize a panel of carbohydrates. While Race 1 is endemic to the Southern United States, Ralstonia solancearum race 3 biovar 2 (R3b2) is not known to occur naturally in the U.S. and therefore is considered a select agent. R3b2 can devastate crops, leading to major economic losses worldwide: global damages in world potato crops alone from R3b2 are estimated to be over $950 million per year, which is why its quarantine is crucial for protecting American agricultural interests. Presently, visual inspections remain the primary way to monitor agricultural imports for this disease. Asymptomatic plant material, however, easily could allow R3b2 to breach U.S. borders. There have been, in fact, at least two recent cases of R3b2 infestations being traced back to geranium imports from Kenya and Guatemala, exemplifying the need for an early detection system to be in place at U.S. ports of entry. Available tests are not fast enough to conduct large-scale inspections, nor are they sensitive enough to detect asymptomatic plant material. In addition, most of the tests require dedicated laboratories able to conduct complex assay procedures. In the recovery plan for Ralstonia solancearum race 3 biovar 2, which was drafted as part of the National Plant Disease Recovery System, the USDA lists one of the "Highly important" research priorities as: "explor[ing] novel diagnostic techniques to detect R3b2 quickly and at low concentrations, such as ... highly sensitive antibody-responsive cell culture systems." PathSensors, Inc. has responded to this research priority by proposing to build a multi-well diagnostic device using CANARY® technology.CANARY® technology is a cell-based biosensor technology that demonstrates unprecedented performance in terms of speed, sensitivity, and ease-of-use when compared with other standard detection technologies. The USDA has invested over one million dollars to develop a CANARY® biosensor cell line to detect Ralstonia and the USDA CPHST Laboratory in Beltsville, MD, has used this biosensor to achieve one of the lowest limits of detection of any CANARY® biosensor based, or other type, assay for the detection of Ralstonia, achieving an LoD of 3 cfu from infected potato slices. PathSensors, Inc. intends to add its expertise to the USDA's efforts to develop efficient sample preparation methods to achieve similar results with geranium stem samples. Instrumentation and sample processing steps need to be optimized, however, in order for CANARY® technology to be used efficiently at plant testing stations. To maximize the benefits of CANARY® technology, we must develop a new multiplex instrument platform as well as a simple and rapid means for plant sample preparation and introduction to the biosensors. The instrument system will consist of commercially available parts linked together and controlled through an integrated computer software system. Once the optimum sample process steps have been established, the user will be led through all steps of the assay via a computer software program. Positive or negative sample identifications will be provided by processing the CANARY® light output signal through an automated detection algorithm.Upon its completion, this system can be deployed and used in survey programs, field labs, and diagnostic testing facilities. Moreover, this system will be made available to exporters to assure their plants are pathogen-free prior to shipping. After developing a multi-well diagnostic device, PathSensors, Inc. will further adapt the system for use with other existing plant pathogen CANARY® cell lines, such as those for Phytophthora