Validation of an Alternative Small Stem Assay for Blight Resistance in Chestnut Seedlings and Recommendations for Broader Use

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnut trees (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4- to 5-mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting. Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we showed that mean orange zone canker length (OZCL) and a multifactor principal components analysis-based blight resistance index gave results consistent with predictions derived from two methods of blight resistance phenotyping and percentage of American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnut trees, mean OZCL of backcross families ranged from intermediate (F₁ hybrid-level) to low (wild-type American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and postinoculation stem dieback was not apparently related to the stem tip inoculations. Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding. Thus, the AltSSA can prevent time, resources, and orchard space from being used on susceptible trees.