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...