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Koch's Postulates

Activation of isolates was successful with almost all isolates so I am now able to move on to carry out Kochs postulates with them. The aim of Koch's postulates is to confirm a diagnosis that a particular suspected organism is causing the observed disease. The steps were first described by Robert Koch in 1882 and later added to by Erwin F Smith in 1905. The steps are as follows:

1. determine the suspected pathogen is consistently associated with the disease
2. isolate the suspected pathogen from the diseased plant and grow it in pure culture on artificial media and describe it.
3. Inoculate the isolated organism onto a healthy plant of the same species to see if it causes the same disease symptoms.
4. Re-culture the pathogen and determine that it is the same as the original isolate.

Steps one and two have already been completed so I am now going to work on steps 3 and 4.

Isolate activation

I am hoping to 'activate' some isolates by inoculating them into rhododendrons and then re-isolating them from the plant after infection has occurred in an attempt to remind the isolates how to be virulent. Some times we have isolates that have been maintained for so long in culture that they start to behave strangely or they stop sporulating for example. This is a technique to re-activate them before using them in experiments.

Soil inoculation relative success

I have been trying to inoculate rhododendron roots with Phytophthora spp for a while and Kim and I have tried several methods that are reported in the literature. I set my latest trial up in the mist chamber to maximise the water availability and finally I seem to have symptoms all be it in only one rep out of three and on only one species out of 7 but never the less I have symptoms yay. See the wilting plant on the right compared with happy plant on the left.

Detached leaf experiment

I ran another repeat of my detached leaf inoculation where I dip inoculate leaves in zoospores suspension and incubate them without wounding for 2 weeks. I spent 8 hours on Labor Day doing this and it looks as though it hasn't worked. This is the fourth time I have set this experiment up and the other 3 times I got good data, although the trends don't exactly match what I expected at least the leaves got infected. This time I was meticulous so I can't figure out why only 9 leaves out of 150 developed lesions. Gah! At least this shows the expected trend but there are so many misses I can't really use this as quantitative data. Perhaps I can just note the observations. So disheartening.

twitter

In case you haven't already noticed I have joined twitter and I'm having my tweets posted to the gadget in the right column of this blog so if you want to get an insight into what I am doing on a daily basis this will contain little snippets. You can follow me on twitter - @claret84

New photos of farm inoculation

are available to view through Picasa as a slide show on my photography blog.

Plants inoculated in the mist chamber

P. citricola
P. citrophthora

P. syringae

P. cinnamomi

P. nicotianae

googly eyes

I posted a little video at the bottom of this page, just for fun. It is a sketch from Saturday Night Live. The site that hosts the video streams an advert before the video begins. It's pretty funny I think.

beautiful drip spread lesions

Here are some new photos from today in the mist chamber, the Phytophthoras are doing nicely in there where it is all wet.

Current Activities

I'm helping to compile data from a study looking at a potential new species of Phytophthora, and conducting kochs postulates on several isolates in collaboration with another lab. I'm also pulling together the data from my poster to put into a paper written by a post doc in our lab to try to get that published too. I'm running a mist chamber inoculation concurrently with my field work to compare findings at 2 different temperatures under very moist conditions. I will also be repeating a detache leaf inoculation using P. ramorum as I obtained rather strange results last time.

that's all for now

APS meeting

Here I am at the American Phytopathological Society Meeting in Portland OR earlier this week. I presented this poster on the pathogenic fitness of the clonal lineages of Phytophthora ramorum.

Disease spread

The first photo shows P. syringae spreading aerially, probably by rain splash during winter. The second photo shows P. nicotianae spreading within the plant during late spring/early summer.

research in the news

Here is an article from February of last year on the Salem news website, talking about the importance of research on Phytophthora spp. and other pathogens for the nursery industry in Oregon which recently exceeded $1bn in sales value. Several people I work with are mentioned and my project on the epidemiology of Phytophthora spp on Rhododendron is mentioned.

Field experiment

Finally I am having some success at inoculating my field experiment. Here is a graph showing my initial data. There are significant differences between species and between cultivars and as expected P. syringae (7) is the most aggressive pathogen so far. This photo shows a Rhododendron inoculated with Phytophthora syringae as part of my field experiment. You can see how the infection has spread aerially by rain splash to near by leaves and has dripped down on to the two leaves below the inoculated leaf.

trip to the state capitol

On Friday I went to the capitol building in Salem to testify in favour of house bill 2508 for the abolition of fees for graduate students with a tuition waiver. I went along with other members of the Coalition of Graduate Employees (CGE) the union for the grads on OSU's campus, and the GTFF which is the equivalent at the University of Oregon. The GTFF and CGE are part of the larger union, the American Federation of Teachers (AFT).

the proposed HB 2508 would stop the universities in Oregon from charging fees on top of tuition to graduate students when we have funding which provides a tuition waiver. The current situation sees 17% of my net pay go back to the university each year, last year this amounted to $2840.

Here is a photo of members of CGE and the GTFF in front of the capitol after testifying for over an hour to a concerned committee.
It was a great experience and it seemed that they committee really took on board our message although in these economic times of severe budget cuts it seems unlikely that any dramatic changes will occur in the near future but hopefully this will begin to open up conversations about how we can improve the situation for graduate students in Oregon.

The CGE has also posted remarks about Friday's trip on their blog and you can download an audio recording of the proceedings from Friday's hearing here. The hearing for HB 2508 begins 16min 29sec into the recording and I testify at 38min 15sec and it closes at 1hr 36min 26sec after 12 testimonies in favour of the bill and 1 agains the bill (someone sent to represent the university).

Statistics and Abstract

I've been analysing the data obtained from the experiments last term where I dipped whole plants in inoculum and measured the number of lesions and the area of the lesions on the plant. I have submitted an abstract for a poster to present at one or two meetings this summer.

Phytophthora ramorum - pathogenic fitness of the three clonal lineages

Clare Elliott, Virginia McDonald and Niklaus Grunwald

The Oomycete pathogen Phytophthora ramorum causes sudden oak death on oak and ramorum blight on a wide range of ornamental plants causing severe economic losses to the nursery industry. The US population of P. ramorum consists of three distinct clonal lineages referred to as NA1, NA2, and EU1. The hypothesis of differences in pathogenic fitness among these three lineages was tested through the infection of detached leaves and whole plants in wounded and un-wounded inoculations of Rhododendron. In independent experiments the fitness of isolates within lineages was determined using the fitness components lesion area (LA), sporulation capacity (SC), incubation period (IP_w) and the area under the lesion expansion curve (AULEC) on two cultivars of Rhododendron; the more susceptible cv. R. catawbiense ‘Boursault’ and the moderately resistant cv. ‘Lee’s Dark Purple’. For all wounded detached leaf experiments and for the whole plant work 3 isolates from each clonal lineage were tested, and for the un-wounded detached leaf assay the sample size was increased to 10 isolates per lineage. In the non-wounded whole plant experiments incidence was measured by number of infection points and number of leaves infected and severity was measured by total lesion area and average lesion area per leaf. LA demonstrated significant differences among lineages in two out of three wounded detached leaf experiments; however, SC, IP_w and AULEC showed no consistent significant differences among lineages. There was also no consistent significant cultivar by lineage interaction among the wound inoculated experiments. The non-wounded whole plant dip inoculations showed a trend towards a difference between the NA1 lineage and EU1 and NA2 (0.1>p>0.05) but variability among isolates within lineages means that these slight differences are not statistically significant. In one out of the two experiments on whole plants significant differences between isolates within lineages were observed (p<0.026). style="">

This study indicates that there are minor differences in fitness components among P. ramorum clonal lineages but suggests also that within lineage variability is great. Experiments are ongoing with an increased sample size of 10 isolates per clonal lineage.

Troubleshooting

I am having a lot of trouble getting my rhododendron leaves to become infected and having discussed the issues with a group of scientists working on Phytophthoras I have come up with a list of ideas which may be the reason for this:

1. The plants are dormant and have suberised for the winter and so their innate immunity afforded by their thick cuticle is especially effective.
2. The plants have been wounded by me cutting leaves from them for previous experiments and this has elicited systemic aquired resistance to infection.
3. The plants at the farm are irrigated by water which has been treated and this may be inhibiting the infectivity of the plants.

To test these theories I will carry out a number of small scale experiments using detached leaves to determine if there is an effect of wounding the plants or if there is a difference in infectivity year round or how the irrigation is affecting the plants.

back to the research

Well it's a new term and I'm not taking any classes and I'm not teaching so I can devote all my time to my research and it's the perfect time for it as I am having my first committee meeting this term and I will give my research proposal seminar at the start of next term too so I will have plenty of time to prepare fully for those two events.

I have finally got my committee members together and set a date so I need to prepare a summary of my research proposal for them and get my program of study signed off by them. I am gearing up to set up another farm experiment with a slightly different foliar inoculation method and I'm making plans for a lot of other little experiments to supplement the main project.

Yesterday I spent the day helping Kim take down her soil inoculation experiment in the growth chamber and although her experiment didn't go as planned it was a good experience for me as I will need to follow a similar method when I take down my farm experiment. There are definately some lessons to be learnt from these initial runs of the soil inoculation and this might mean tweaking my experimental design for my field experiments a little. We'll see.

Guppies

So it's not research related but I had my first batch of guppy babies at long last. Hurrah!