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.

Summer Class

I'm now all done with classes for this term, I have no final exams and I've finished my term project for statistics. I have almost 3 weeks off from classes before the summer term starts so I will be able to get some good work done on the research front without homework and classes getting in the way. This summer I'm taking a class called Plant Disease Diagnosis with 11 Botany students. It looks like a great class, it's all field trips and lab analysis. Assignments will be unknown diseased plant samples and we will have to diagnose the diseases. We are going all over Oregon to visit all kinds of crops from Christmas trees to mint farms with vegetables and fruits in between. For more details on the fun trips I'll be going on check out this website for the class and click on the Syllabus link. It will be particularly good since all the class trips and labs will be on Thursdays and Friday mornings so the remainder of the week will be free of interruptions.

Plants are coming

Finally I have received confirmation that 2 orders for rhodies have gone through so I expect to have the first batch next week and I'll be able to begin inoculating. Hurrah! This first experiment will be a little different from those I have been working on for the field as I will be using dip inoculation which will result in multiple infection sites over the whole plant and I will be working in the quarantine chamber for this too so I am getting smaller plants to fit more in. The result will hopefully be extremely sick plants within 10 days so that I can then detach some leaves and measure the lesion area on them. The plant is to get this little dip experiment underway and mostly done this month and then begin the first farm experiment some time in July. I'm not sure how this will work out but since classes finish this week I will have more time to work on it.