Bacteriophages as a Biocontrol agent of Soft Rot in Potatoes
Hodson, Trevor
Faculty Mentor: Don Breakwell, Microbiology and Molecular Biology
Introduction
Pectobacterium carotovorum (Pcc) is a principle causative agent of soft rot in potatoes. It causes losses of up to 60% in potato yields in the USA (Mantsebo et. Al, 2014) and approximately $50-$100 million dollars’ worth of losses yearly in multiple types of crops. Because potatoes grow beneath the soil surface, there is no way of treating the potatoes until they are harvested. Even then, no postharvest methods of controlling this pathogen exist (Wood et. Al, 2013). A spray-able bacteriophage cocktail specific to Pcc could save millions of dollars-worth of crops each year by minimizing soft rot.
Active Pcc infection is regulated by a quorum sensing mechanism, which means there needs to be a certain minimum concentration of bacteria in order for infection to develop (Denis Faure, 2007). A bacteriophage cocktail specific to Pcc would only need to keep bacterial levels below the required concentration to prevent soft rot. Our goal was to develop such a cocktail.
Methodology
P. Carotovorum is an infectious plant pathogen, so Dr. Breakwell first had to work with Dr. Grose to receive a permit to work with them through the EPA. A strain of Pcc was shipped from the American Type Culture Collection (ATCC). In order to test the host range of Pcc phage later on, we needed to isolate several strains.
Pcc secretes pectolytic enzymes that enable it to use pectin as a food source, this is a moderately selectable attribute so we poured two kinds of pectin based plates, DL-CVP and CVP mediums, that had been used by researchers previously to isolate Pcc (B. S. Bdliya, 2004; Perombelon, 1991). We collected rotten potato and tomato samples from BYU off-campus housing apartments. We inoculated nutrient broth (Difco) with pieces of the rotten samples. Samples were homogenized using a metal probe and left to incubate overnight. We then used a disposable loop to streak for pure culture on the CVP and DL-CVP mediums.
Pcc also grows on nutrient agar while some of its fellow pectolytic bacteria do not, so I poured nutrient agar plates and streaked the colonies that had grown on the CVP and DL-CVP onto nutrient agar plates. We took the strains that grew on nutrient agar and pectin-based media and froze them down. To confirm we had Pcc, I isolated the DNA of a few strains using a Powersoil DNA isolation kit (MO BIO, Inc.) following the protocol provided (MO BIO Inc., 2015). We amplified the 16S rRNA gene using PCR, and purified the product using the Ultraclean PCR Clean-up kit (MO BIO, Inc.). We submitted the genomes for sequencing and used BLASTn to compare the sequences with GenBank. One strain was Pcc so we moved forward to isolating phages, but we also continued to work on isolating more strains of Pcc.
To isolate bacteriophages, I created a dual-layered plate infused with P. carotovorum that I then used to test phage enrichments for plaque formation. The bottom layer in the plates
was nutrient agar; the top agar was half-strength agar (7 g nutrient agar per liter) containing 1 g CaCO3 per liter. While still liquid, I mixed 4.5 ml of top agar with 0.5 ml of Pcc culture prior to plating it on the nutrient agar base.
For the phage enrichments, I took a 250 ml Erlenmeyer flask and added 50 ml nutrient broth, 2 g CaCO3, 50 g of a soil sample taken from a potato garden and 3 ml of an 18-hour old Pcc nutrient broth culture. I incubated the culture overnight and then took 5 ml aliquots and centrifuged them at 6000 x g for 5 minutes. The supernatant was decanted and 0.5 ml of chloroform was added to the solution to lyse some of the cells in order to free potential phage within the cells. I then vortexed and centrifuged the mixture at 6000 x g for 5 more minutes. I removed the supernatant and spotted 12 ul of the supernatant on the dual-layer plates. I then incubate at 25oC and checked for plaque formation, an indication of phage infection. This process was repeated many times with various modifications in an attempt to get plaque formation, but have not yet been successful.
Results
20 strains were isolated on the CVP, DL-CVP media. 16 of those strains also grew on nutrient agar. One of those 16 strains has been identified as Pcc and named Pcc1. As mentioned previously we have been unable to get any plaque formation, however, using the above phage isolation protocol. The protocol was modified using different strength agars to see if the agar concentration was the inhibitory factor but still no plaques formed.
We noticed that we were not getting a very well defined bacterial lawn on top of the dual-layer plate and wondered if the lack of bacterial growth in general was masking the presence of bacteriophage plaques. We tried incubating the plates at different temperatures with no success. LB plates were used as a bottom layer for the dual-layer plate to see if that would help to grow a more defined bacterial lawn but it did not. We are now investigating different mediums for the dual-layered plate to get clearer plaque formation.
Discussion
We know that we can successfully isolate P. Carotovorum strains from soil samples and we are continuing to do so, however, this task is meaningless unless we are able to perfect phase 2 of our project, isolating phages using those strains as bait.
The greatest difficulty so far in our protocol for isolating phages is getting Pcc1 to grow a thick bacterial lawn on top of our dual-layered plates when mixed with liquid nutrient agar so that we can easily identify plaques. This suggests that if we can just find the right medium make-up for our dual-layered plate, a medium that will produce a thick Pcc1 bacterial lawn, phage isolation can quickly proceed.
Conclusion
Isolation of P. Carotovorum phages remains an important area of further research. Their successful isolation and incorporation into a phage cocktail could save millions of dollars in potato crop losses yearly. Although progress has been made, these phage as of yet remain elusive.