Plant seeks phage: Searching for viruses that can protect plants from bacterial disease
By Bryden Fields, Research Associate on the Ralstonia Phage project
Bacteriophages (phages) are viruses that infect and kill bacteria. Phages can have very specific host ranges and we can harness this specificity to kill certain bacterial strains whilst leaving other bacteria unharmed. In the Ralstonia Phage project, we are searching for phages that can be used to target a pathogenic bacterial strain, called Ralstonia solanacearum. This bacterium causes bacterial wilt disease in a wide range of plant species, including tomatoes and potatoes. In this post, we’ll tell you how we’ve been searching for, identifying, and trialling novel UK phages for use as potential biocontrol agents of bacterial wilt disease.
Fishing for phages
Firstly, we needed to find some phages that could infect and kill R. solanacearum. The best place to look was in locations where we were likely to find R. solanacearum. In the UK, this means taking some samples of river water which is where R. solanacearum can be found during the warmer months of the year. We find the bacterium in river water because it can asymptomatically infect woody nightshade, a wild plant host that grows on riverbanks. Interestingly, previous outbreaks of bacterial wilt in the UK have been traced back to river waters used for irrigating potato fields. To take samples we just collected some river water in a tube.
Enrichment and purification of phages
Back in the lab, we added up to six different R. solanacearum strains into the river samples and incubated them for several days. This process, called enrichment, allowed Ralstonia specific phages in the river water to multiply to increase abundance. We then spotted a drop of each enriched river sample onto an agar plates containing one of our six Ralstonia strains. If a circular ‘clearing zones’ appears around the drop it indicates that the river sample contains at least one phage that can kill that R. solanacearum strain. Re-plating this river sample on a new Ralstonia agar lawn is a way of diluting the sample so that any clearing zones are made by individual phage particles. A single clearing zone made by a single phage can then be cut out of the agar and regrown on a fresh plate to produce a purified phage sample.
A photoshoot for our phage friends
It is not possible to identify what type of phage we have isolated just by looking at the phage clearing zones alone, so the next step was to isolate DNA from our phage and send it off for genome sequencing. While waiting for the sequencing results to come back, we could quickly classify the phage we had found by imaging our phages using transmission electron microscopy. We found that our UK river phages have a wide range of different morphologies (shapes). This meant we could classify them into several bacteriophage types, including phages from myoviridae, podoviridae and Siphoviridae families.
Testing, testing…
Having identified phages that can infect R. solanacearum in lab conditions, we next test whether the phage can successfully infect R. solanacearum in the rhizosphere soil and prevent bacterial wilt of tomato plants. We run these tests in large scale glasshouse trials at Fera. These trials involve inoculating tomato plants with combinations of both R. solanacearum and phage. Over a couple of weeks, we monitor the plants to see if they develop wilting symptoms or are protected from disease by the presence of the phage. These trials are still ongoing but once we have found our winning phage treatment, we will start testing whether we can mass produce it for use in agriculture.
To find out more about this research please visit the Ralstonia Phage project page. You can also watch this short talk given by project lead Ville Friman.