Term is almost over

There is less than one week remaining of classes and then only finals week. I'm flying back to the UK on December 12th. I have to run the last of the plant pathology labs today and then the lab final on Thursday. Next week is the plant path final which I will help to grade and then my final exam for Forest insect and disease management, and I also have to complete and turn in my collections of insects and forest pathogens which are almost complete, just need to label them.

lack of progress

I've had a horrid cold all week and so I have not been as productive as usual, in addition this week, the class I TA for turned in two pieces of homework for me to mark as well as completing their second midterm exam so I have spent a lot of time grading and not much time in the lab doing research. I've also been working on ordering merchandise such as t-shirts and tote bags for the Botany and Plant Pathology department to buy in time for Christmas and hopefully make a few pennies for the the graduate student association (GSA) of BPP to pay for student travel grants and coffee meetings! Plus I have a midterm on Monday in forest disease management so I am studying a little for that this weekend, and I can't wait for Thanksgiving next weekend and a nice long break away from the normal routine. The term is flying by, and I just can't wait for next term when I will not have to take any classes, and will be able to devote all my time to research and actually get things done! On the other had there is so much to do before the end of term and it is approaching fast...

About turn

Well, the second of my detached leaf inoculations had an equally high failure rate to the first try despite doubling the concentration of the inoculum and meticulous set up and monitoring. I have come to the conclusion that this may be due to the state of the plants I took the leaves from, I guess they are just heading into winter and the leaves are thickened and suberised, and although the preliminary experiment I did in early September to trial the method had a 100% success rate this has not worked in the full scale set up I began in October. It's rather frustrating. I will now try dipping whole leaves to see what happens, if this still doesn't work I may just have to put this off until the growing season begins next year.

In other projects I am trying to make inoculum for the farm experiment but that is proving tricky once again, even though I thought I had nailed a method which would work. Never mind. I am going to trial an agar inoculation method now to see if that might be more reliable. Since for this research the objective is to study the epidemic not the infection process so in theory it shouldn't make much difference how I inoculate so long as infection occurs.

Busy

This term has been so busy I have not had time to update this site with new posts. I am enjoying teaching the labs for plant pathology and I am hoping to do this again next Fall to fulfill the teaching requirements for the PhD program.

I am halfway through the Forest Insect and Disease Management class that I am taking and I've completed the insect collection for it. I now have to make a pathogen collection for the second half of term.

In other news Curtis and I got engaged on the 14th of October, our 3 year anniversary so we now have to make plans for our wedding with will probably (not definately) happen next summer in the UK.

I am trying hard to keep my research projects moving forward despite having little time leftto devote to them after teaching and taking a 5 credit class. I have some time data for the whole plant dip experiments I was doing so that just needs analysing. I am now working on a detached leaf experiment and then on Friday I will hopefully set up another foliar inoculation at the farm with help from some helpful people in my lab. Being so busy I have been particularly appreciating the assistance of my colleagues especailly Kim and two undergraduate workers, Rob and Jamie. I am luck to work in a lab where so much support is available.

I am also starting to coordinate my graduate studies committee so that they can approve my program of study. I have asked three professors who have all said yes so I'm almost set to have my first meeting with them. These are also the people who will ultimately examine me to determine if I should pass with a PhD.

I am going home to England for just over 2 weeks at Christmas so that will be a nice holiday for me.

Zoospore Release

Here is a photo I took today looking through the microscope at sporangia releasing their last remaining zoospores. You can see one squeezing through the opening of the sporangium. The flagellated zoospores inside the sporangia were swimming around quickly looking for the way out before zooming off at great speed.

The new term!

I haven't had much to post here recently as I took a 2 week break to show my family around Oregon. It was a great time and I'm pretty sure I impressed them with the wildlife and geology of the region.

Now the new term has begun and I have a rather hectic schedule. I'm going to be a teaching assistant (TA) for introductory plant pathology this term. I'm also taking a 5 credit class in Forest insect and disease management which promises to be a lot of work but very interesting.

Somewhere I will find time to do some research too...

Soil Inoculation Assessment

Yesterday I assessed my soil inoculated plants and noticed some Phytophthora symptoms, however it has only been 2 weeks and it seems to me to be rather quick for such advanced symptoms to develop. Looking closer at this example in the annotated photo you can see the flagged branch with wilted, necrotic, and curled leaves, and the branch itself is also very dark in colour and necrotic. I feel that it is possible that these plants were not free from infection with Phytophthora when I received them from the grower and now that they are in my irrigated plot the conditions are ideal for the disease to develop. If you look closely it looks as thought a branch has been removed, possibly due to infection, to make the plants look good for sale but the inoculum was still present and has re-emerged in the wet conditions I have put the plants in. Alternatively, it is possible (and preferred) that the pathogen I inoculate the soil with (P. cactorum) has been splashed up onto this lower branch and infected it, or that the roots have become infected and thus cut water supply off to this first branch, causing it to wilt and die (expressing foliar symptoms caused by a root problem). In order to determine what is going on here I will have to try to isolate the pathogen from the margins of this necrotic tissue and grow it in culture to sequence it and identify what species it is. If it is what I applied to the soil then this is great news, if not it is contamination and not good news at all.

A plant with no chlorophyll

Monotropa uniflora commonly known as Indian pipe is a plant which has no chlorophyll. I saw this one in the forests near Mount Hood, Oregon. Since it can't photosynthesise without chlorophyll it acts as a parasite to fungi which are in symbiosis with trees and so it can get it's energy and food indirectly from a photosynthesising tree through a mycorrizal fungus which grows with the roots of that tree. These plants are commonly associated with American beech and Pine trees through Russula or Lactarius mushrooms. It's flowers are pollinated by bees. Information found at this website. Also see this wiki.

Oregon Wilderness Photos

Scroll to the bottom of this page to see a new slide show of photos taken last Friday (August 29th) when Curtis and I camped by the Salmon River and hiked up the Salmon Huckleberry trail.

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.

working in the containment hood with a broken hand

I am able to carry out most tasks even if a little slower than usual. Here is how I managed to fit an extra large latex glove on my cast, using tape to hold the spare fingers out of the way! My co-workers found it rather funny.

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!

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:

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!

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.

Farm Experiment Site

Here are some photos I took yesterday as the farm site is finally set up for the first experiment to go ahead. There is a shade cloth overhead to keep the plants from getting sun scorch, and also a very nice irrigation system has just been installed. You can see the mist created by the irrigation, which keeps the leaves nice and susceptible to disease! The different cultivars and treatments in the experiment are randomised among each rack of plants.

In this picture you can see the shade cloth overhead and the complicated plumbing for the 6 zones of the structure so that I can program each zone (which are all colour coded!) to come on for a certain time separately!
This is a rain gauge, one of many sensors I have in the plot to record the conditions the plants are experiencing. the rain is funnelled into a small tipping bucket which holds a specific amount, each tip is recorded and the water drips out of the bottom of the bucket.

Fish

Above are my first male and female betta fish or siamese fighting fish, unfortunately the male died soon after I got him and I replaced him with a new one and also replaced the female for fear that she too was sick with the same problem which killed the male. I got an almost Identical pair and tried to mate them but I couldn't get them to do it, the male would make a bubble nest but then they didn't ever put eggs in it. In the end the female died, I don't know what of, I know the water chemistry is fine and I'm feeding them bloodworms which they love. Anyway I am bored with Bettas, they are not that interesting and have to be kept in separate bowls which is annoying. Below is my original male.

This is my newer male who I still have. There is also a trap door snail in here too on the bottom. He slides around the glass keeping it clean!
In a larger bowl I have 2 red capped gold fish and a black moor fish with bulging eyes (see bottom photo):

coming soon

meet my fish - I will soon be posting photos of my new fish and possibly of some plants I am cultivating to turn my room into a mini jungle!

soil inoculation and farm experiments - I will be getting my farm Rhodies next week and I am currently making some infested soil inoculum for root inoculations at the farm in August.

music - I recently found this website which customizes your listening to your music tastes, check it out. pandora radio

Oregon Coast Photos

Photos from the Oregon Coast are now at the bottom of this page.
To solve some of the discussions that have been going on the 7th picture shows a tree stump probably washed up as drift wood, and the 8th picture shows a sand beetle or some kind of crustacean.

Optimising Zoospore Concentration - Statistics Project continued

Continued from Optimising Zoospore concentration post
Sorry that not all the graphs and figures are included.

Results:
Nested Analysis of Variance on Square Root Transformed Data: Linear model:
Y_ijkl = μ + τ_i + α_j + β_k(j) + e_ijkl Where Y_ijkl is the observation, α_j is the fixed effect of the i^th species, β_k(j) is the fixed effect of the j^th level isolate nested within species, τ_i is the fixed effect of treatment and e_ijkl is the random experimental error. i = 1, 2, 3, 4, j = 1, 2, 3, k = 1, 2, 3 (the 3 isolates for each species are different so there are a total of 9 isolates, 3 nested in each of the 3 species).

Conclusions

The analysis of variance clearly showed that there was a significant difference in the amount of zoospores obtained between the four different treatments (F_(3,24)=42.3, p<0.001)>(2,24)=6.75, p=0.005) and between the isolates nested within species (F_(6,24)=9.01, p<0.001). The 5mL treatment with SPW or n/s soil extract was identified as being the best treatment, yielding the highest concentration of zoospores to volume of inoculum produced (Table 4). There was no significant difference between the remaining three treatments (Figure 5).

No further analysis of the differences between species and isolates were carried out as these differences were not of interest to the research being undertaken. It may be interesting for future studies to investigate the extent of any significant differences between isolates of the same species; however this did not pertain to the research question for this study.

My aim for this experiment was to optimise the concentration of zoospores produced from a single Petri plate culture. These results show that the best treatment, out of the 4 treatments tested herein, was the 5mL application of SPW or n/s soil extract as this yielded the highest ratio of concentration of zoospores to volume of inoculum obtained. In answer to the research question posed; reducing the volume of SPW or n/s soil extract applied to the broth cultures does increase the concentration to volume ratio of zoospore inoculum obtained.

I will now be able to obtain the concentration of zoospore inoculum that I require for all of the eight Phytophthora spp. that I am working with. Previously P. citricola and P. cambivora had consistently been poor producers of zoospores and I was unable to achieve the desired concentration of inoculum for my plant inoculation experiments. The results of this experiment lead me to conclude that I should treat these species with only 5mL of SPW or n/s soil extract (respectively) rather than 10mL which I had used previously.

I am unable to suggest a biological reason for this stark difference between the 5mL treatment and the 10, 15 and 20mL treatments. The purpose of treating the broth cultures with this SPW or n/s soil extract is to induce sporangia (which release zoospores) production through starvation conditions and so I can only assume that for the species studied herein the reduction of this rinse volume to 5mL enhances the effect of the starvation conditions and promotes greater sporangia or zoospores production. It is unclear whether the changes in zoospore concentration result from greater sporangia production or increased zoospores produced per sporangia. It seems particularly interesting, and unexplained, however that there are no significant differences between the other three treatments so that the relationship between treatment volume and response is not linear. Further research into the factors involved in this response must be undertaken in order to ascertain the nature and cause of the relationship.

Containment Inoculations

The following photos are of me inoculating plants in the containment chamber.

Here I am preparing the inoculum to a specified concentration and then dipping the whole small plants in the inoculum.


Here I am suited and booted with my plants which I bag up after dipping to keep them in a moist environment which is most conducive to establishment of infection.

Optimising Zoospore Production in Phytophthora Species

This is what I did my statistics term project on:

In the course of my studies of the epidemiology of Phytophthora species on Rhododendrons I need to produce inoculum with which to infect the host plants. Phytophthora spp. are Oomycete plant pathogens, also known as water moulds. The inoculum produced comes from structures called sporangia, borne from the vegetative hyphae of the Phytophthora spp. cultured in vitro. These sporangia release motile zoospores which are the asexual propagules which infect the host tissue.

The process of producing zoospore inoculum involves inoculating a liquid broth media (V8 100) with the Phytophthora isolate, rinsing the 5 day old culture with distilled water to remove the V8 100 broth, and replace it with a volume of either filter sterilised pond water (SPW) or non sterile filtered soil (n/s soil) extract to induce sporangia production by creating starvation stress conditions (Pettitt et al. 2002, Ahonsi et al. 2007).

I am working with 3 isolates of each of 8 species of Phytophthora and I have found that different species produce zoospores at different concentrations. For the inoculations of host plants I must standardise the concentrations of zoospore inoculum produced to 10,000 zoospores mL^-1. I have found that two Phytophthora spp. consistently produce half the inoculum concentration of the other species such that I am unable to dilute the inoculum to 10,000 zoospores mL^-1(because you cannot increase the concentration, you can only dilute it). I aim to optimise the concentration of zoospores produced from a single Petri plate culture.

One way to try to do this is to adjust the volume of SPW or n/s soil extract applied to induce sporangia production to see what effect this may have on the zoospore inoculum produced. Obviously just decreasing the volume of liquid in the plate will increase the concentration of zoospores in that volume of liquid, however it is possible that either increasing or decreasing the volume of SPW or n/s soil extract applied to the cultures may alter the stress conditions to encourage increased sporangia production thus improving the concentration to volume ratio of inoculum obtained.

References:
Pettitt, T.R. et al., Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water. Plant Pathology, 2002. 51(6): p. 720-727.
Ahonsi, M.O., Banko T.J., and Hong C., A simple in-vitro `wet-plate' method for mass production of Phytophthora nicotianae zoospores and factors influencing zoospore production.70(3): p. 557-560. Journal of Microbiological Methods, 2007.

Research question: Does reducing the volume of SPW or n/s soil extract applied to induce sporangia production increase the concentration to volume ratio of zoospore inoculum obtained?

Research objective: Determine the volume of SPW or n/s soil extract to use to maximise the number of zoospores produced.

Experimental Design: This experiment will be a nested design with 4 levels of treatment (5, 10, 15, 20 mL; volume of SPW or n/s soil extract) applied to 9 isolates of Phytophthora, 3 nested within each of 3 Phytophthora spp. Each isolate acts as a replicate for treatment so that r = 9. The experimental unit is one isolate growing in a Petri dish. The response variable will be the ratio of the concentration of zoospores to the volume of inoculum yielded from the method described above. MINITAB and MS Excel software will be used to conduct an Analysis of Variance (ANOVA) and a multiple comparisons test to determine the best treatment.

Results to follow...

Photos

As you can see I have posted a slide show above of a trip Curtis and I went on yesterday to the coast. I have also put a slide show of Oregon spring flowers in the photography section of this blog (see links to the left). Enjoy!

Inoculations

this first photo (above) shows my plants in the midst of the inoculation process
the second picture (below) shows the different growth morphologies of three isolates from the same species, it's difficult to see in the picture how dramatic the differences are, but if you click on it the picture will enlarge and you can see that the left plate is a faster growing flat isolates and the right plate is slower growing and the top and right plates have a raised fluffy morphology.
This last picture (below) shows my little plants outside post inoculation, they look like little Christmas trees with all those colourful clips, but sadly they are just waiting to die.

Today I inoculated 9 plants with my Phytophthora spp as another practice run before the field experiments begin. I tried two different methods; zoospores and agar, to see which works best and to determine whether it really matters which method I use. Here are a few photos of the inoculuation in progress.