Shima Chaudhary,

I think that the best way to quantify Phytophthora is through zoospore inoculum production. Please see Byrt and Grant (1979) method (Byrt, Pauline, and B. R. Grant. "Some conditions governing zoospore production in axenic cultures of Phytophthora cinnamomi Rands." Australian Journal of Botany 27.2 (1979): 103-115.). The number of zoospores/mL could be determined with a bright line haemocytometer. To reisolate Phytophthora from roots or from the soil I advise you to use a baiting method, as described in Jung T, Blaschke H, Neumann P, 1996. Isolation, identification and pathogenicity of Phytophthora species from declining oak stands. European Journal Forest Pathology. 26, 253–272. Good luck!

Hi Carmen,

Thanks for replying, I wanted to grow seedlings of sour orange in soil with known propagules. I though adding a certain known number of chlamydospore per known volume of soil can make it accurate. I can add zoospore suspension with known count, as well. Another test I want to do is quantify using real time PCR technique and see if adding more propagules to the soil correlates with real time PCR values. I thought adding known number of chlamydospore is a better idea.

Hi Shima,

Sounds interesting! Once Phytophthora infects roots through zoospores, the relationship between inoculum and root response could be better than number of chlamydospore. How do you ensure the number of chlamydospores which would germinate? Moreover, in practical terms I believe that it´s easier have a zoospore suspension with concentration determined than isolate chlamydospore. Unfortenatly, I don´t know how to separate chlamydospore, maybe someone else could be help you better. Good luck!

Hi Shima,

most researchers indeed use zoospores for inoculation of aerial plant parts. However, for inoculating soil and/or roots chlamydospores are a good option, because they are more robust. I'm attaching a method we're using at NORS-DUC for producing P. ramorum chlamydospores for soil inoculation. Hope it helps.

good luck,

Wolfgang

Hi Shima,

For inoculating roots or soil, chlamydospores could be prepared as described in Sánchez et al. 2002 (Phytophthora disease of Quercus ilex in southwestern Spain. Forest Pathology, 32, 5–18) for Phytophthora cinnamoni or Serrano et al., 2012 (Susceptibility to common herbaceous crops to Phytophthora cinnamomi and its influence on Quercus root rot in rangelands. Eur J Plant Pathol 134. 409-414) if you prefer use zoospores. Both are very easy and effective methods. You can quantify the spore concentration using a Neubauer chamber.

Good luck!

María

Hi all,

Thanks for sending me some protocol to isolate and quantify chlamydospores. I will test and let you guys know, as to how it goes. I am planning to use a known value of zoospore suspension to surface infect roots and may be reinoculate with a syringe. But, to add to soil, chlamydospore appeared to be more controlled. Also to create different concentration of propagule in soil and quantify by real time PCR, chlamydospore appear to be better.

Appreciate the feedback.

Shima