Methods
Summary
Surface sterilized seeds will be grown in pasteurized soil. During initial growth, half of the young seedlings will be inoculated with a spore solution of the candidate endophyte. Once the seedlings have grown at least two true leaves, all will be challenged by inoculation of an agar plug with growing Cryphonectria parasitica into a scalpel incision on each seedling stem.
Differences in disease outcome between the endophyte inoculated seedlings and the endophyte free seedlings will be evaluated with a student t-test to determine if the endophytes significantly protected the seedlings.
Challenges
Potential challenges include both logistical and experimental concerns. Logistically, a research partner located east of the Mississippi river due to pathogen quarantine concerns (as Brandon, the primary researcher, currently lives in Oregon).
Experimentally, the inability to eliminate pre-existing endophytes harbored within chestnut seeds, and fulfilling Koch's postulate by re-isolating the candidate endophyte from inoculated seedlings will be the most challenging to overcome. Regardless, endophyte inoculation should increase blight resistance among seedlings regardless of pre-existing any endophyte load; otherwise its utility in the field would be of dubious value.
Pre Analysis Plan
The hypothesis to be tested is that preinoculation with the biocontrol candidate endophyte will increase survival, reduce lesion damage, and/or lengthen time until death compared to untreated seedlings once challenged with the blight fungus.
The seedling experiment is designed so that any effect size should be large. If the endophyte is able to protect the seedling, clear and significant differences should be seen.
Protocols
Browse the protocols that are part of the experimental methods.