Evaluating the Effect of Temperature on the Development of Xanthomonas fragariae, the causal agent of Bacterial Angular Leaf Spot
Bill Turechek & John Hartung
Progress Report
The objectives are addressed in the order they were presented in the original proposal.
1) Determine the effect of temperature on in vitro growth rates of Xanthomonas fragariae.
The motivation behind objective 1 was to help us develop a set of reasonable treatments to address objective 3. Figure 1 shows the average growth of two strains of X. fragariae. Growth occurs most rapidly within the range of 16-28 C and occurs much more slowly at temperatures of 10 C and less. We attempted to measure growth at higher temperatures, e.g., 34 C, but struggled with contamination of the cultures. In fact, it took a number of attempts to generate these curves. The reason being was that the liquid cultures were sampled repeatedly to measure growth and, unfortunately, contaminants were introduced in the process. This could largely be attributed to the inexperience and lackadaisical attitude of the technician that worked on the project; he no longer works for me. Nonetheless, the initial results clearly showed that X. fragariae grew at very low temperatures and, thus, one could speculate that infection, or at least the development of symptoms, would also occur at these temperatures (see Objective 3).
I am in the process of hiring a new technician (the applications are in!) and I plan to continue these simple experiments with the 4 genotypic strains of the bacteria to determine if any differences exist among the 4 strains.
2) Determine the potential of hot-water treatment for eradicating X. fragariae from strawberry nursery stock.
To measure the effect of hot water treatment on bacterial growth, we proposed to use a protocol that called for serial dilutions of bacterial cultures after exposure to their heat treatment. This proved to be an exceptionally laborious procedure, fraught with some of the same labor issues as discussed above. Upon reconsideration it occurred to us that, practically speaking, a heat treatment either works or it doesn’t. Therefore, the protocol was modified where 1 ml aliquots of bacteria dispensed in eppendorf tubes were heat treated and a 5 µl subsample was spotted on suitable growth media. Five days later the presence or absence of growth was recorded. We used this protocol to evaluate 6 temperatures and 12 exposure times on 4 genotypic strains of X. fragariae. The results of 6 runs are shown in Figure 2 below. At 52 and 56 C, complete kill was attained with only one exception; greater then 90% kill was consistently attained at 44 and 48 C. The cells that made it through these latter two heat treatments appeared to be “heat tolerant”. The heat tolerant colonies have different cultural characteristics than “normal” colonies, particularly lacking the characteristic yellow ooze. These colonies, however, test positive as X. fragariae using PCR. It is possible that these surviving, heat-tolerant colonies may be less virulent, or not pathogenic at all, to strawberry given that certain pathogenicity factors are associated with the bacterial ooze. This needs to be tested.
The bottom line is that exposure to 44 C for 2 hr may be a sufficient heat treatment for strawberry if the only cells surviving are non-pathogenic. This is important because our preliminary work with strawberry plants, as well as work by Buchner, shows that strawberry will not survive heat treatment for a prolonged period of time at 48 C or greater. In contrast, we have had plants survive heat treatment up to 8 hr at temperatures above 42 C. We did much preliminary work this summer heat treating plants, and we have it narrowed down a set of treatments that we will be testing on ‘Camarosa’ and ‘Diamonte’ as soon as the plants arrive in January.
3) Determine the effects of temperature and leaf wetness on the development of angular leaf spot and their role in the systemic invasion of the pathogen.
We haven’t gotten very far with this objective. This is due to a number of reasons. First, our growth chambers have been malfunctioning on a number of levels. Luckily, we have solved most of these problems. Second, it has been difficult to obtain consistent infection with our inoculation technique. However, the recent publication by Hildebrand et al. (Can. J. Plant Pathol., 2005, 27:16-24) has shed light onto what we may have been doing wrong. Unfortunately, their publication addresses some of the work that we were planning on doing for this objective, specifically, looking at the effects of temperature on disease development. They did not, however, address systemic movement. We are planning to continue this work once the chambers are fully functional and my technician settles in.
Addendum: One of the most significant research projects I am working on developed directly from the needs of this project. In short, we are developing a real-time PCR protocol for detecting viable cells of X. fragariae. Standard PCR is a very sensitive molecular tool that can be used for specific detection of DNA from an organism of interest. The problem, however, is that the procedure does not discriminate whether the DNA originated from viable or dead cells. We have adopted a method from the recent literature where this is possible and have gotten it to work with the DNA primers for X. fragariae developed by Pooler and Hartung (1995). Furthermore, we’ve created 3 new sets of primers and fluorescent probes and have developed a real-time PCR procedure. This not only allows us to discriminate between viable and dead cells, but permits us to quantify how many cells of each there are. In the context of our work, this procedure will be very useful for evaluating the efficacy of heat treatment on nursery plants, and will be very useful for monitoring the systemic movement of bacteria in plants. I have a student visiting from Davis for three months on an internship that will help us fine tune the protocol so that it may be used reliably. More on this when I resubmit the grant…
Figure 1. Growth of X. fragariae in sucrose-peptone liquid media. Growth was measured with a spectrophotometer twice a day. The maximum value for absorbance is 2.
Figure 2. The proportion of X. fragariae colonies surviving heat treatment. Each point represents the average of four genotypic strains over six experimental runs (24 observations). Time is represented on a logarithmic scale to better show the results of shorter duration treatments. The inset numbers represent the temperature treatment (i.e., 56, 52, 48, 44, 40 and 36 C). Note: the 1 minute treatment represents the 0 minute control, i.e., no neat treatment (the log of zero is undefined).