BLIGHT RESISTANCE TESTING

⇓Presentation slides from our Annual Meeting by Leslie Bost 

OCF Annual Meeting 2018

Detatched Leaf Assay

  • Leslie Bost
  • Colton Zirkle

OCF Breeding Program | Blight Screening

 At a glance: What We Need

Our Ozark chinquapin restoration projects would be expedited by an early blight screening assay.

An ideal early screening assay would be: simple, repeatable, nondestructive to the tree, quantitative, and able to detect intermediate & high levels of resistance. 

Blight Screening

Stem Inoculation Method

Traditional screening involves inoculating stems of >3 year old trees with the blight fungus (C. parasitica), then measuring resulting cankers months later

Destructive to host trees

Detached Leaf Assay

Involves inoculating detatched leaves with the blight fungus and measuring resulting necrotic lesions 5-10 days later

Leaves can be collected from few-month-old seedlings or fully mature trees

Results are measured after less than a week

  •  Experimental leaf assay results correlate well with stem assay results

Isolating the blight

Blight cultures isolated from SW Missouri

A sample of bark is taken from an actively growing blight canker on a chinquapin tree and placed in a petri dish where the fungus can grow. Then a small sample of the leading edge of the growth is placed in a new petri dish where the isolated fungus will continue to grow and be used for our inoculations. 

AC and CC seedlings are grown-out before we begin our study so young leaves can be collected 

»Chinese Chestnut (CC)

low susceptible control

»American Chestnut (AC) 

highly susceptible control

Why do we use controls in our study? 

If the assay can can resolve differences between species that have known levels of high and low resistance to the blight, results of the study are more reliable

CIMG3078
American chestnut
20181111_082704
Chinese chestnut
20180802_193134
Ozark chinquapin

Select genetic lines of Ozark chinquapin growing on our research plots will have some of their leaves removed and inoculated with the blight (along with AC and CC leaves) 

OVERVIEW

RAPID LEAF ASSAY

In spring of this year the OCF will begin an important study which will put us years ahead in our restoration efforts allowing us to quickly identify the most blight resistant trees for our breeding program.

Measuring the necrotic area at the inoculation site

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 collecting pollen from large, surviving trees and cross breeding those trees for resistance.  Seeds collected from selective breeding have been planted in test plots across the tree’s native range and many are now producing seeds.  We are now at the point that we need to evaluate the blight resistance of these selectively bred trees.

Traditional methods for testing blight resistance involve inoculating the stems of trees that are greater than 3 years old with active, virulent blight. The tests last several months and can either harm or kill the trees.  However, the OCF has recently partnered with the American Chestnut Research and Restoration Project at SUNY College of Environmental Science and Forestry who have pioneered new methods to evaluate resistance in transgenic chestnuts.   The new method requires leaf inoculation, is non-destructive, and only takes 7-10 days for quantitative results.

This study will begin spring 2018.  Trees at test plots will be selected and prioritized for testing based on lineage and overall health.  Young leaves will be removed from the trees and transported back into a sterile lab space.  They will then be cleaned, inoculated with the blight, and allowed to incubate.  The blight fungus will cause a necrotic area to develop around the inoculation site on the leaf (see photo).  After a period of 7-10 days the area of the necrosis will be measured.  Leaves from American and Chinese chestnut will also be inoculated with the blight to serve as controls in the study.  Chinese chestnuts are resistant to chestnut blight and inoculated leaves should show small or nondetectable necrotic areas.  Whereas, American chestnuts are highly susceptible to the disease and should develop large necroses.  We expect our Ozark chinquapin to fall somewhere in the middle.  As the OCF’s work progresses, this study will serve as a meter to determine if our breeding projects are developing trees that are more resistant to the blight.