Latest Blight Resistance Screening & Updates (Oxalate Leaf Disk Assay)

New Blight Resistance Testing

Oxalic Acid Assay:  Screening Method To Evaluate Resistance 

by Leslie Bost Carter  |  published 2021

Screening to Evaluate Resistance of Trees Developed on Research Plots 

The mission of the foundation is to restore blight resistant Ozark chinquapin to its historical range.  For the last 10+ years, we have worked toward this goal by cross pollinating resistant  Ozark chinquapin to develop seed with improved blight resistance.  Seeds collected from selective breeding have been planted in test plots across the tree’s native range and many are now producing seed. 

In 2020 the OCF established the first Ozark chinquapin Restoration Planting in Carroll county Arkansas.  We plan to continue releasing  blight resistant trees, selectively bred for local adaption to microsites in the Ozark chinquapins natural range.  

In summer of this year the OCF did a study to identify the most blight resistant trees for our breeding program and restoration plantings. We recently adopted a new method to evaluate resistance.  The new method is easier than leaf assays that require detached leaves to be inoculated with the blight–and takes less time for results. It’s called an exogenous oxalic acid leaf disc assay, or, oxalic acid assay, for short.    

Photo: Leaf samples collected from research plots in Missouri and Arkansas

How an Oxalic Acid Assay Works

The fungal pathogen responsible for the blight, Cryphonectria parasitica, secretes oxalic acid (oxalate) and kills healthy tissue which forms cankers on the bark of susceptible trees. Oxalic acid assays rapidly screen for relative resistance based on the percent of living tissue remaining on leaf discs soaked in oxalic acid for 24 hours.  Because the leaf discs are uniform in size, living tissue can be quantified easily with image software. Instead of detaching & inoculating leaves with chestnut blight, this assay requires us to simply expose leaf tissue directly to oxalate and observe their tolerance.  The assay is rapid, effective, and is often used to evaluate resistance in genetically modified (transgenic) plants with inserted genes that detoxify oxalic acid produced by fungal pathogens.

Methods

I was supplied leaf samples from Ozark chinquapin trees located on an OCF research plot in Missouri and the new Arkansas Restoration Planting; and from those samples, selected 10 chinquapin of interest to screen. Pure Chinese chestnut (C. mollissima) was used as a resistant control and a susceptible Ozark chinquapin (tree number ’46’) was selected for a moderate-susceptible control.  The blight testing we performed in 2019 showed this tree had the least resistance compared to the other Ozark chinquapin screened in that study. 

Genetic variation is responsible for differences in resistance levels between full sibling families of trees. This is something we wanted to explore. 

For every tree, 8 leaf discs of uniform size were punched out using a #3 cork borer and then soaked in 50mM oxalic acid for 24 hours. Photographs of the leaf discs were uploaded to plant disease quantification software and analyzed. This software calculates the exact percent of healthy tissue on each leaf disc. The mean (average) percent of living tissue was calculated and plotted on a bar graph (see attached photo).

Results

We have a full sibling family of “10X” trees we are particularly interested in because we would like to understand which of them has the highest resistance. Previous blight screening (2019, Leslie Bost) demonstrated a variability in resistance among these siblings. Based on the leaf disk experiments I performed, tree number “10X^6’’ appears to have more resistance relative to the other 10X trees screened in my assay (see bar graph).

Tree number “36XX – 10X LH-12“ has the highest amount of living tissue after soaking in oxalate compared to every tree in the assay, and should be a priority for additional screening and future selection as a parent tree for breeding. This tree is growing on our Lucky Hollow Restoration Planting in Northwest Arkansas and is very disease resistant. The tree is a cross made from two parent trees that were both also controlled crosses.

I’m looking forward to seeing the restoration potential these trees offer!