Todd Jarvis and Julianne Grose, Micro and Molecular Biology
Fire Blight, caused by the bacteria Erwinia amylovora, is a highly contagious fruit tree disease that is difficult to treat. This disease causes a tree to whither away, as if it had been burned. It is estimated that Fire Blight causes more than a 100 million dollar loss in agricultural goods each year1. In an attempt to treat fire blight and prevent it from spreading, large doses of antibiotics are sprayed on infected trees. These antibiotics do little in the way of helping to treat the trees. In addition, the antibiotic run off from the treatment finds its way into our underground water systems and sewer lines. This in turn may fuel the development of antibiotic resistant bacteria that have recently begun to emerge. Our project is a unique approach to treating Fire Blight. Our hypothesis is that multiple phage exist that can act as effective Fire Blight cures, and, unlike antibiotics, the phage we are pursuing will be organic Erwinia killers that are harmless to humans and are biodegradable. Achieving our goal will bring solutions to both the problem and the previous failed attempts to solve it.
The work of our research team has resulted in 15 isolated phage collected from various samples. My project for the last year has been focused on characterizing these species and developing an effective treatment model. Characterizing the phage has included many different types of studies. First we needed to determine that the phage we had isolated would only infect Erwinia amylovora. This involved performing infection tests with a large number of bacterium closely related to Erwinia. Next we isolate the DNA of each phage, sequenced it using BYU’s 454 sequencer, and then annotated each genome to determine the proteins each phage DNA contains. Finally our research team had sought a new way to test Erwinia infection and treatment with phage in a laboratory setting. This involved developing an effective way to grow seedlings, several tests to determine appropriate infection doses, and some preliminary treatment with our phage.
Our last year of research has yielded many exciting discoveries. Through initial DNA extraction of our isolated phage we determined that we have 11 Erwinia phage. Our annotation efforts have confirmed that each of these 11 phage are unique. In order for our phage to be effective for treatment they need to be of the lytic lifestyle and specific only for Fire Blight. Through our studies we discovered that 9 of our phage are lytic, while 2 are lysogenic. In addition, host infection experiments found that each of these phage are specific only for our three Erwinia amylovora strains, and not other closely related bacteria. Characterizing these phage has helped us to predetermine their effectiveness as treatment agents before performing any tests.
Furthermore, in an attempt to develop a new model of testing, I have focused on creating an effective in-lab test for the treatment of Fire Blight. I have developed a method to grow new apple seedlings in a matter of weeks. After perfecting this process, I tested different methods of infecting the seedlings with the Erwinia bacteria at various titers. Through this process we have found an appropriate infection model that will allow us to test the phage we have discovered without wasting adult trees, thousands of dollars, and exorbitant amounts of time.
The results of our research over the last year have been nothing less than what we have expected and anticipated. Our phage were able to rescue 100% of the apple seedlings from Fire Blight, while 100% of those infected died. Further, developing an effective infection model will allow us to perform many preliminary tests with various combinations, or phage cocktails, of our nine lytic phage. This will allow us to determine which phage can be used for further studies on infected orchards.
Erwinia amylovora continues to infect many trees each year, and with the ban of antibiotics, our research is becoming more and more important. Over the next six months we will be accelerating our research so that our phage cocktails will be ready for testing in orchards in the spring of 2015. This will involve utilizing our newly developed infection model to test different combinations of our phage isolates and studying the protein products of each phage to determine which specific genome sequences characterize a highly lytic activity. We anticipate that these trials will continue to yield the expected results and that our treatment will be ready for use in orchards soon.
1Norelli, J. L., A. L. Jones, and H. S. Aldwinckle. (2003) Fire Blight Management in the Twenty-First Century: Using New Technologies that Enhance Host Resistance in Apple. Plant Disease 87 (7): 756-765.