I have isolated a fungi species from the trunk of a tree species and I want to test whether this is the causal pathogen that created a canker on that tree stem. I am working on polybag seedlings and is confused about how to inoculate the seedlings.
As far as I understood you want to inoculate the seedling with the fungi you have isolated, is it so? if yes, then you have to firstly grow your isolated fungus over Mycological Broth (Fungal Broth) incubated under 25-27 degree Celsius for 48 hours. Now after incubation centrifuge this culture broth at 10000 rpm at room temperature for 10minutes. Discard the supernatant and dissolve the pellet into autoclaved normal saline. Now irrigate the roots of seedlings with equal amount of this prepared solution to check the pathogenic efficacy of your isolate.
Vagmi Singh Thank you for your response. Can you please elaborate on the irrigation process. Should I use a bucket where the inoculated saline solution will be poured and the seedlings will be placed after removing from polybag soil? And If that is the process how will I maintain the seedlings to observe for pathogenic efficacy?
Speaking as someone who reviews a lot of papers that include pathogenicity tests, I have to ask if this is a pathogenicity test, or something else? It is tricky to use seedlings to prove a symptom seen on mature trees. You could do a stem inoculation, but to do that, you would almost certainly want to create a wound on the stem (like using a cork borer to make a hole) and then add the inoculum. Evaluating a resulting canker will be tricky, because it is such an unnatural system (young plant with wounded trunk). You would need to use a positive and two negative controls. One negative control would be inoculation with water (if you used a spore suspension to inoculate the pathogen) or an agar plug (if you used a culture plug to inoculate the pathogen). The other negative control would be a fungus known not to cause cankers on that host. The positive control would be a pathogen KNOWN to cause cankers on that host. After you incubate the inoculated trees, you would probably be measuring the length of the resulting lesion. The tricky part is that even with the no-inoculum control, there may be a lesion. With the nonpathogen control, there might be a lesion. So how do you evaluate the lesion produced by your test organism? How long is its lesion compared to the known pathogen? If it is comparable, you can suggest that your unknown can cause cankers, and I will more-or-less believe you. But if the lesion length is closer to the one caused by the nonpathogen, I will more-or-less disbelieve you.
Kouadri Mohamed El Amine is suggesting a detached stem pathogenicity test, and as a reviewer I would take a very dim view to that unless you had the proper positive and negative controls. Detached stems are, by definition, stressed plant tissue, and stressed plants behave differently than non-stressed plants. If you had the right controls, however, I would probably be OK.
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