Leaf Assay Results
Screening Restoration Ozark Chinquapin For Resistance
by Leslie Bost Carter
Introduction
In the spring of 2019 we completed a series of leaf assays to evaluate the trees in our breeding program and identify individuals with the most resistance to chestnut blight. We have hundreds of trees on our research plots located in 3 states, and the trees on those plots represent the best genetics from across the native range of the Ozark chinquapin. There are three types of trees we have at these various plots: Parent trees, resistant crosses (pure inter-crosses), and clones.
Our parent trees are individuals that were grown from the seed of rare Ozark chinquapin trees with natural resistance to chestnut blight. Chestnuts are self-incompatible and need another tree in order to reproduce. Just like humans, the progeny share genetics from both parents. There is a chance the seedlings from those resistant parent will have resistance too, but not all of them will because the donor pollen of surrounding trees are from susceptible Ozark chinquapin (Figure 1).
The crosses on our plots are the result of manual pollination of two individual resistant trees. These controlled pollinations allow us to combine genetics of two trees. Female flowering parts are bagged to prevent competing pollen from interfering (Figure 2). We out-plant the resulting seed on our test plots. Those trees are “crosses”. As you you’ll see in a moment, the pure crosses we developed have some of the highest genetic resistance to the fungus.
Figure 1.) A 9 year old Ozark chinquapin grown from the seed of a resistant parent
Figure 2.) Manual cross pollination of a resistant parent trees
The third kind of tree is one of the most important on our plots; clones. These trees are clonal grafts with the exact genetics of the tree they were taken from. Instead of seed, we collected twigs (scion) from these rare resistant trees and grafted them onto root-stock of another tree. These are especially important to our breeding program. These clones can produce reliably resistant progeny when they are bred with other resistant trees. They are particularly valuable to us for enriching the genetics of locally adapted populations (Figure 3).
All of these pure Ozark chinquapin trees have been growing on our plots for the last 9 years, openly pollinating and producing seed. This is the seed we mail OCF members every year.
Figure 3.) Clone of a large blight resistant Ozark chinquapin from private pro McDonald county Missouri.
Traditional Breeding for Resistance To Chestnut Blight
Phenotyping plants for resistance means selecting a tree based on its physical characteristics that show the tree is resistant/resisting the blight. In other words, we find a tree (or a few trees) in an area where chestnut blight has destroyed most of the population, and these are the ones who survived. Over evolutionary time these are the trees whose progeny would continue on, and this natural selection is nature’s own built-in breeding program. Through our genetic resistance breeding program we are helping along a process that would take hundreds of years to happen on its own in nature. All of this is done from the most ecologically sound approach, which encourages the continual evolution of both the tree and the fungal pathogen. We want to ensure the trees have enough resistance and genetic diversity so they’re able to handle any future threats.
Resistance in chestnuts is quantitative. In other words, there is not just one gene responsible for resistance but rather a combination of multiple genes. Studies of the nuclear and chloroplast DNA of Castanea revealed the Ozark chinquapin has the most genetic diversity compared to all North American chestnut species (Dane et al., 1999.) We are taking advantage of this diversity in hopes of capturing those rare alleles.
This method of utilizing natural resistance through a traditional tree improvement breeding program has been successful for a number of other native species threatened by exotic pathogens. Our approach to restore the Ozark chinquapin requires more effort and money than the alternatives but is the most promising path. Genetic resistance is the most important defense a plant has against a fungal pathogen and often the only solution for durable long-term success.
Leaf Assay Material and Methods
As described earlier, our program involves selecting and breeding trees. The next critical step is evaluating those trees for resistance. We screen them by means of artificial inoculation. A detached leaf assay is an experiment where we detach leaves from a tree, inoculate them with a plug of chestnut blight grown in culture, and measure the area of resulting brown necrotic (dead) tissue on the leaves after several days. The average necrotic area appearing on the leaves gives us an idea about the resistance of the whole tree. A tree whose leaves have a small area of necrosis are probably more resistant than those with a large area of necrosis. Even though Cryphonectria parasitica is not a leaf disease, leaf assay results correlate well with whole plant inoculations. In other words, this is a good way of characterizing a tree’s resistance as an alternative to inoculating the whole plant. Detached leaf assays are commonly used for screening plants for resistance to disease, and this particular protocol for chestnuts was developed by Andy Newhouse from SUNY-ESF in New York. We have partnered with them for training on this protocol and they’ve also provided us the pure American chestnuts we used for controls in our study.
(Figure 4) Inoculations were done with a virulent strain of C. parasitica isolated from bark cankers of infected Ozark chinquapin in NW Arkansas. Cultures were maintained on potato dextrose agar (PDA, Difco) and sub-cutlured 3-5 days before the assays. Before each experiment, 4 mm agar plugs containing C. parasitica were punched out using a sterilized cork borer around the perimeter of the actively growing colony.
Ozark chinquapin leaves were collected from trees growing on our research plots and placed in labeled bags and put in a cooler. Approximately 7-11 leaves (from this years new growth) were detached from each individual tree selected for screening (25 trees). Leaves were also collected from greenhouse grown American chestnuts and Chinese chestnuts . All the leaves were washed in 3 baths of distilled water and mild detergent. After gently blotting dry, the leaves were labeled and numbered. (Figure 5)
Figure 4.) The fungal pathogen responsible for chestnut blight, C. parasitica, was isolated from bark samples and maintained in culture for inoculations
Figure 5.) Leaves were collected from 25 Ozark chinquapin trees at several research plots located in Missouri
Inoculation
Using a marker and ruler, a 5 mm area was delineated on the lower surface of the each leaf on the midvein about 30 mm from the petiole. Using a sterile #11 scalpel, a wound was made approximately half the depth of the midvein tissue at the inoculation site. Then an agar plug containing C. parasitica was carefully placed mycelial side down onto the surface of the wound on the leaf. (Figure 6)
The inoculated leaves were then placed in gasket sealed Sterilite Ultraseal containers lined with slightly damp paper towels and placed in a dark room to incubate at room temperature (25 degree C) for 4-5 days. The resulting brown necrotic tissue that developed around the inoculation site of each leaf was measured in length and width to calculate estimated area. Exact area was also calculated using an image analysis software for plant disease quantification called Assess 2.0. (Figure 7)
Chestnut blight is an exotic pathogen from Asia. Over in Asia where the fungus evolved together with the Asian chestnut species (Chinese and Japanese), selective pressure conferred resistance in those species. But our North American chestnut species had very little defense against this introduced disease, in particular the American chestnut (C. dentata). For this reason, Chinese and American chestnuts were used in my study as controls. Both of those species have known levels of host resistance; low susceptibility in Chinese chestnut and high susceptibility in American chestnut. See table for sources of chestnut seedlings in Figure 8.
Figure 6.) The fungal pathogen responsible for chestnut blight, C. parasitica, was isolated from bark samples and maintained in culture for inoculations.
Figure 7.) Exact area of necrosis was calculated using image analysis software for plant disease quantification called Assess 2.0. Manual measurements were also taken to estimate lesion area.
Figure 8.) Seed source for experiment controls
Results
A total of 286 individual inoculations were performed over several experiments during the spring 2019 growing season. The mean necrotic area of 25 selected Ozark chinquapin, Chinese chestnut and American chestnut were compared to determine relative resistance. The table in Figure 9 displays the results of these experiments.
Results indicate we’re making significant advances in breeding trees that are resistant to blight. In particular, the pure crosses. For each tree we tested the distribution of sample variation from the mean is noted by error bars on the graph. The bars give us an idea about how accurately the mean value represents the data. The error bars represent the standard deviation of the data set. A small bar indicates low spread from the mean, larger bar indicates data are more variable and spread further from the mean.
I chose not to include the results of three Ozark chinquapin trees I screened due to small sample size. Those particular trees are young clonal grafts and had a limited amount of good leaves. Those three trees performed very well in the assay and will be re-screened in the future to better characterize their susceptibility.
Two separate experiments were conducted after the study. In the first experiment, I confirmed C. parasitica was the pathogen responsible for the necrosis observed on inoculated leaves by placing a piece of infected leaf tissue from an inoculated leaf on water agar. Mycelium formed and I was able re-isolate Cryphonectria parasitica and subculture a pure colony to PDA. Morphology in culture confirmed identify of the fungus.
The second experiment I performed was done in order to see if any difference in necrotic area would be observed in leaves detached and inoculated later (after longer periods of time lapsed) vs immediate inoculation after detachment from the tree. I detached leaves from different greenhouse grown chestnuts, stored them in plastic bags moistened with distilled water, recorded the time, and placed them in the fridge. 24 hours later I freshly detached leaves from the exact same plants as before, and inoculated them along with the leaves I had detached 24 hours prior. No significant difference in necrotic area was observed for the ones I detached earlier compared to ones detached right before inoculation.
Figure 9.) Selective breeding has yielded several highly resistant Ozark chinquapin. Screening revealed superior blight resistance of parent trees and 1st and 2nd generation crosses.
Figure 10.) Resistant Chinese chestnut and highly susceptible American chestnut controls are shown in the top yellow box, below are the top resistant Ozark chinquapin restoration trees screened in the experiment.
Discussion
The results of this assay show us a few important things. The first thing we can see from the leaf assay is that the 10 years of patient work and careful selective breeding has yielded several highly resistant Ozark chinquapin progeny. (Figure 10) A third of the OCF restoration trees we evaluated had blight resistant levels similar or better than Chinese chestnut. These results are preliminary and additional screening will be conducted in the future to screen the rest of our trees. We are using the information gained from this study to guide selections for breeding next spring.
Looking Ahead
My final thoughts are an appeal. For thousands of years the Ozark chinquapin has benefited man and wildlife, and it is time for us to return the favor. We want to help bring this species back so the trees can thrive and reproduce again on their own in our forests and woodlands. If you care about these trees and want to help us please consider partnering with us and spreading the word. Many people do not know about this tree and its important role in our ecosystem as a consistent nut producing tree for wildlife. We want to do something before the tree fades from the memory and landscape of the Ozarks forever. We are currently doing only about a tenth of the work we would like to do and our only limiting factor is funding. Given the success we are having so far with our program to develop resistance, we are hopeful that in time we will be able to devote ourselves full time to restoring the Ozark chinquapin.
Acknowledgments
A special thank you to the Missouri Botanical Garden here in St. Louis and their incredible staff who allowed me to use their facilities for this project. They provided me access to their lab space for culturing the bight and room in their greenhouse to propagate the chestnut seedlings I used in my study. Some of the materials I used were donated from them. I am very grateful for the friendships I made through this partnership and the OCF would not have been able to do this without their help. I want to thank Matthew Albrecht, Burgund Brassuner, and Justin Lee. A big thank you to Andy Newhouse of SUNY who provided consultation & pure American chestnuts I used for my study. A special thanks to OCF board member Shawn Smith who helped collect bark samples for me. And lastly, I would like to acknowledge my dad Steve Bost who helped me with hours and hours of work collecting leaf samples from our research plots. He is an inspiration to me and many others. Thank you to our members whose donations helped pay for the supplies I used for these experiments and travel expenses. We used those funds to purchase the special software I used, and the camera needed for the pictures the program analyzes. You are helping us save the Ozark chinquapin!
My contact information:
Leslie Bost Carter
lesliebost@yahoo.com
The Ozark Chinquapin Foundation email: ozarkchinquapininfo@gmail.com
President, Steve Bost:stevebostocf@gmail.com
2021 Blight Resistance Testing–oxalic acid assay