Validation of an alternative small stem assay for blight resistance in backcross hybrid chestnuts (Castanea spp.) and recommendations for its expanded use

Abstract

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnuts (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 show that mean orange zone canker length (OZCL) and a multifactor PCA-based blight resistance index gave results consistent with predictions derived from the blight resistance phenotype (two methods) and percent American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnuts, mean OZCL of backcross families ranged from intermediate (F1-level) to low (American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and post-inoculation deaths were apparently unrelated to the stem tip inoculations (e.g., unrelated basal cankering). 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.