Lab photo
Nik has updated the lab members page on his website so you might like to see who I work with. also we took a lab photo this week and so that just went up on the page too (the reason I am standing sideways compared to everyone else is that I was trying to hide my left hand behind my back so you couldn't see my cast).
Counting Cells
In order to adjust the concentration of zoospores in the inoculum I produce for each species or isolate of Phytophthora that I am working with it is necessary to count the number of zoospores in small samples of the inoculum to estimate the concentration. Currently this is achieved using a haemocytometer:
This is a microscope slide with a very fine grid etched into it and when used with a calibrated cover slip the volume of the liquid sample viewed on one square of the grid is known and thus the concentration of particles counted can be calculated from this. Repeat counts are averaged to decrease the error of the estimate. This is a laborious and eye straining activity especially when there are many samples to be adjusted and a time constraint to adjust the concentrations before spores encyst.This week I have been trying out a new piece of equipment called a 'countess' being developed by Invitrogen, a biotechnology company, I have it on loan for a week for testing and feedback. Here are some images of me trying it out.
I was counting zoospores which are smaller than 10 microns hence the spike in the graph at that size on the x axis. It looks as though there were a couple of sporangia in the sample too as there are 2 small spikes around 40 microns which is about the size of a sporangium which release zoospores (see time lapse slide show in the right bar of this page to see zoospores being released).The Far West Show
Yesterday I went to the far west show in Portland at the convention centre. It is a big trade show for the region for anyone in the nursery industry or horticulture or anything to do with production through retail in the industry. I grabbed several catalogs from nurseries who could potentially supply me with the rhodies I am working with. 
back to the research
this blog is meant to focus on my research so here are some results of my plant dip inoculations from the 9th of August. 'Cultivar' refers to the host plants, and 'lineage' refers to the pathogen isolates (there were 3 isolates used from each lineage), all were Phytophthora ramorum. This graph shows some nice differences so hopefully when I have the results from all three of the experiments I will be able to show some nice statistically significant findings. Cultivar A looks to be less suceptible than cv. B, and lineage 1 seems less virulent/infective/aggressive than lineages 2 and 3.
Garden Harvest
Although we've been picking at the tomatoes for a few weeks now this is the first big collection of produce from Curtis's garden! Yum! There's plenty more to come too.
I broke my hand
I fell off my bike and fractured the base of my pinky and ring fingers of my left hand. Splint for 4 weeks:
My snail had babies!
Apparently these trap door aquatic snails are hermaphroditic, just like garden snails which means that they have both male and female reproductive parts but still have to mate with another snail to reproduce, this means that every snail can have babies, not just females. My snail must have mated prior to me buying it and has now given birth to live young. I only noticed them this morning when I was changing the water and one snail got stuck on the end of the tube I was using to siphon the water up with. They are so small they are about the size of the pebbles, but they still slide around surprisingly fast when they are not hiding in their shells. They can produce up to 8 or so babies, but I may have lost some by not knowing they were there and sucking them up. They take up to 2 years to grow to full size and maturity so it could be a while before I make any more snail babies, however it is also possible for the adult to have saved some of the sperm for a second batch without needing to mate again. We'll see. I'll be on the look out from now on. DISEASE!
I just went to visit the farm and check on things for the first time since the set up as I was rather busy taking down the dip inoculations and I also had to go on a two day field trip to Hermiston.
Here are the first signs of infection since the experiment was set up on Tuesday at the farm! This is great news, because it means that despite the hot conditions and the time it took us to get all the 108 plants inoculated the zoospores survived and infected at least some of the plants!
Checks on the inoculum viability the day after the set up confirmed motile zoospores in at least 5 of the 8 species, and inoculum plated on the selective media PARP grew colonies for 6 of the 8 species. The failures in these two check methods are for different species so overall the 8 species there is at least one confirmation that there were viable infection propagules in the inoculum used the day following the experiment. I.E. for the species which did not grow on PARP there were motile zoospores the following day, and for the ones which were inconclusive when looking for motile zoospores did grow on PARP. This is really encouraging information after all the effort that went into this!
Here are the first signs of infection since the experiment was set up on Tuesday at the farm! This is great news, because it means that despite the hot conditions and the time it took us to get all the 108 plants inoculated the zoospores survived and infected at least some of the plants!
Checks on the inoculum viability the day after the set up confirmed motile zoospores in at least 5 of the 8 species, and inoculum plated on the selective media PARP grew colonies for 6 of the 8 species. The failures in these two check methods are for different species so overall the 8 species there is at least one confirmation that there were viable infection propagules in the inoculum used the day following the experiment. I.E. for the species which did not grow on PARP there were motile zoospores the following day, and for the ones which were inconclusive when looking for motile zoospores did grow on PARP. This is really encouraging information after all the effort that went into this! Assessing disease on dip inoculated plants
Here are some photos of the process of assessing the plants I dip inoculated with P. ramorum isolates in the containment chamber.
I cut the infected leaves from the plant and lay them on this blue sheet and I photograph them next to a label I printed to identify the treatment and repetition number. I also include a penny for scale.
I then cut all the remaining healthy leaves from the same plant and lay them out in the same way. This is so that I can measure both the total plant leaf area and the total area of diseased tissue.
The end result:
I cut the infected leaves from the plant and lay them on this blue sheet and I photograph them next to a label I printed to identify the treatment and repetition number. I also include a penny for scale. 
I then cut all the remaining healthy leaves from the same plant and lay them out in the same way. This is so that I can measure both the total plant leaf area and the total area of diseased tissue. 
The end result: 
Map of shade structure
This is the layout of the shade structure where I am working for my field experiments at the North farm. The current experiment is being conducted in the right bottom and right middle sections (zones 1 and 3). Zone 6 illustrates the pattern of irrigation. Click on the image to enlarge it if you can't read the text. 
Plant Pathology in the News
It's not a pathogen that I am working on but this is a good example to illustrate the importance of research in plant pathology. Here is a news article from the BBC about a new wave of bacterial infections on horse chestnut trees in the UK caused by Pseudomonas syringae pv. aesculi. The article offers little information about the pathogen or why it has suddenly become a problem. Confusingly the article mentions two other problems facing these trees, an insect pest (moths from Greece) and a fungal pathogen causing leaf blotch, which are separate problems but may be interlinked with the outbreak. For example it is possible the bacteria was introduced by an insect vector or that the moths and fungal infections rendered the tree more susceptible to the bacterial infection either through stress or wounds.
I am currently taking a plant disease diagnosis class in which we have been learning how to systematically diagnose plant problems and their causes. Each week we are given an unknown plant sample and we are to fill in a diagnosis form for it. This involves identifying the host plant, understanding the pattern of the problem on the plant or plant part and in the environment around the plant such as the whole field or wherever the plant was collected from. Then looking at the symptoms and signs of any pathogens or pests or clues to environmental or human issues causing the problems.
One of my samples in this class was a branch of a cherry tree with severe gumming (bleeding cankers). I sucessfully isolated Pseudomonas syringae pv. syringae from a canker and cultured it on Kings B agar. This media provides specific nutrients which Pseudomonas species can utilise to make a pigment which glows under UV light. This is a rather simple and fun diagnostic tool for diseases caused by P. syringae.
This disease on horse chestnuts is of interest as it bears resemblance to epidemics of Dutch Elm Disease, Eastern Filbert (Hazelnut) Blight and Sudden Oak Death which are other diseases which also threaten or have devastated trees populations over wide areas in the past. Management of plant diseases on forest hosts such as these trees is rather different to management of diseases on annual crops for example. The epidemiology of these pathogens varies according to factors of the disease triangle (HOST-PATHOGEN-ENVIRONMENT). For disease to occur there must be a virulent pathogen, a susceptible host and conducive environmental conditions. The spread of this epidemic on horse chestnuts will depend on the mode of pathogen spread, the availability of susceptible host trees and management practices in use.
I could go on about the history of the tree diseases I mentioned and the importance of understanding the pathogens and their mode of spread and the conditions they require but I will save that for another day. I'll be taking a forest insect and disease management class in due course and also a forest pathology class so then I'll be bursting with more fascinating stories to tell you!
I am currently taking a plant disease diagnosis class in which we have been learning how to systematically diagnose plant problems and their causes. Each week we are given an unknown plant sample and we are to fill in a diagnosis form for it. This involves identifying the host plant, understanding the pattern of the problem on the plant or plant part and in the environment around the plant such as the whole field or wherever the plant was collected from. Then looking at the symptoms and signs of any pathogens or pests or clues to environmental or human issues causing the problems.
One of my samples in this class was a branch of a cherry tree with severe gumming (bleeding cankers). I sucessfully isolated Pseudomonas syringae pv. syringae from a canker and cultured it on Kings B agar. This media provides specific nutrients which Pseudomonas species can utilise to make a pigment which glows under UV light. This is a rather simple and fun diagnostic tool for diseases caused by P. syringae.
This disease on horse chestnuts is of interest as it bears resemblance to epidemics of Dutch Elm Disease, Eastern Filbert (Hazelnut) Blight and Sudden Oak Death which are other diseases which also threaten or have devastated trees populations over wide areas in the past. Management of plant diseases on forest hosts such as these trees is rather different to management of diseases on annual crops for example. The epidemiology of these pathogens varies according to factors of the disease triangle (HOST-PATHOGEN-ENVIRONMENT). For disease to occur there must be a virulent pathogen, a susceptible host and conducive environmental conditions. The spread of this epidemic on horse chestnuts will depend on the mode of pathogen spread, the availability of susceptible host trees and management practices in use.
I could go on about the history of the tree diseases I mentioned and the importance of understanding the pathogens and their mode of spread and the conditions they require but I will save that for another day. I'll be taking a forest insect and disease management class in due course and also a forest pathology class so then I'll be bursting with more fascinating stories to tell you!
the first foliar inoculation
Kenny and Kim helped me with the set up which is far too much for one person to do in a day. We set up a little mobile lab bench at the farm to prepare the inoculation caps. 
Nik Grunwald visited us while we were setting up, to see how it was going and he took this photo of me inoculating one plant. 
Here is how we worked at the table preparing the caps with inoculum and cotton wool. 
Here is the end result, hundreds of plants covered in multicoloured caps and clips. I hope some of them get sick. Now I'm off on a field trip to Hermiston, OR, camping overnight there as part of my plant disease diagnosis class. This weekend I will be analysing the data from the first dip inoculation experiment in the containment chamber so photos of that will be here shortly.
The big day
Today is the big day for the set up of my first full scale foliar inoculation field experiment. This experiment is designed to run each season of the year for 2 years to allow for epidemiological data to be compared between the 8 different Phytophthora spp being studied, the 2 Rhodie cultivars being used, and the 4 seasons of the year. I will also be running a concurrent root inoculation experiment in the same way which should begin in the next couple of weeks. Pictures coming soon...
Soil inoculum
Here are the jars of inoculum I am preparing to use to inoculate the soil half of the experiment at the farm.
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